Peptide fragments from filoviruses and their uses

ABSTRACT

Isolated peptides comprising one or more antigenic sites of filovirus glycoprotein and methods of their use and production are disclosed. Nucleic acid molecules encoding the peptides are also provided. In several embodiments, the peptides can be used to induce an immune response to filovirus glycoprotein, such as Zaire ebolavirus glycoprotein, in a subject, for example, to treat or prevent infection of the subject with the virus.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/414,960, filed Oct. 31, 2016, which is incorporated by reference inits entirety.

FIELD

The present disclosure relates to immunogenic compositions comprisingone or more peptide fragments from filoviruses that can induce an immuneresponse in a mammal.

BACKGROUND

The 2014 epidemic of highly pathogenic ebolavirus in Western Africaresulted in tens of thousands of infections and deaths. With occasionalsmall outbreaks of new cases in West Africa and the possibility oflong-term persistence of virus in some survivors, it is feared thatfuture outbreaks can occur, resulting in severe epidemics. Developmentof an effective vaccine against ebolavirus is a high priority, both forpre-epidemic preparedness and for rapid vaccination to control futureoutbreaks.

Protection against Ebola Virus Disease is, at least, partiallyattributed to the humoral immune response, since passive transfer ofantibodies to naïve non-human primates can protect the recipientsagainst lethal ebolavirus challenge. However, no single assay has beenfound to be predictive of protection while the correlation of antibodytiters measured by various assays has not been clearly demonstrated.Further, the difficulty of conducting adequate randomized controlledtrials to demonstrate vaccine effectiveness impedes vaccine development.

SUMMARY

Disclosed herein are isolated peptide fragments of filovirusglycoprotein (GP) that induce an immune response to the GP thatneutralizes infection by the virus. The peptide fragments include anidentified antigenic site of the filovirus GP that is capable ofinducing a neutralizing immune response in a subject. Accordingly,isolated peptides are provided that contain a disclosed filovirus GPantigenic site. In some embodiments, the isolated peptide is from anebolavirus GP and induces an immune response to ebolavirus GP thatneutralizes subsequent infection with the ebolavirus. The peptide can befurther conjugated to a carrier to facilitate presentation to the immunesystem.

In some embodiments, an isolated peptide is provided, wherein thepeptide comprises, consists essentially of, or consists of the aminoacid sequence set forth as the antigenic site VI of a filovirus GP, suchas SEQ ID NO: 9: KNITDKIX₁QIIHDFX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWW, wherein X₁ isD or N, X₂ is V or I, X₃ is K or N, X₄ is T, P, or N, X₅ is D or N, X₆is G, T, D, or N, X₇ is D or N, X₈ is N, D, or G, and X₉ is D or S,wherein the peptide is no more than 100 (such as no more than 75 or nomore than 50) amino acids in length and induces a neutralizing immuneresponse to filovirus (such as ebolavirus) in a subject. For example, anisolated peptide comprising, consisting essentially of, or consisting ofthe amino acid sequence set forth any one of SEQ ID NOs: 10-13 or 53 or4, 14-16, or 54.

In some embodiments, an isolated peptide is provided, wherein thepeptide comprises, consists essentially of, or consists of the aminoacid sequence set forth as the antigenic site VI.1 of a filovirus GP,such as SEQ ID NO: 6: FX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWWT, wherein X₂ is V or I,X₃ is K or N, X₄ is T, P, or N, X₅ is D or N; X₆ is G, T, D, or N; X₇ isD or N, X₈ is N, D, or G; and X₉ is D or S, wherein the peptide is nomore than 100 (such as no more than 75 or no more than 50) amino acidsin length and induces a neutralizing immune response to filovirus (suchas ebolavirus) in a subject. For example, an isolated peptidecomprising, consisting essentially of, or consisting of the amino acidsequence set forth as any one of SEQ ID NO: 5 or residues 14-30 of anyone of SEQ ID NOs: 14-16 or 54.

In some embodiments, an isolated peptide is provided, wherein thepeptide comprises, consists essentially of, or consists of the aminoacid sequence set forth as the antigenic site V.7 of a filovirus GP,such as any one of SEQ ID NOs: 3, 32-34, or 55, wherein the peptide isno more than 100 (such as no more than 75 or no more than 50) aminoacids in length and induces a neutralizing immune response to filovirus(such as ebolavirus) in a subject.

In some embodiments, an isolated peptide is provided, wherein thepeptide comprises, consists essentially of, or consists of the aminoacid sequence set forth as the antigenic site V.6 of a filovirus GP,such as any one of SEQ ID NOs: 2, 26-28, or 56, or 43-46 or 57, whereinthe peptide is no more than 100 (such as no more than 75 or no more than50) amino acids in length and induces a neutralizing immune response tofilovirus (such as ebolavirus) in a subject.

In some embodiments, an isolated peptide is provided, wherein thepeptide comprises, consists essentially of, or consists of the aminoacid sequence set forth as the antigenic site IV.1 of a filovirus GP,such as any one of SEQ ID NOs: 17-20 or 58, wherein the peptide is nomore than 100 (such as no more than 75 or no more than 50) amino acidsin length and induces a neutralizing immune response to filovirus (suchas ebolavirus) in a subject.

In some embodiments, an isolated peptide is provided, wherein thepeptide comprises, consists essentially of, or consists of the aminoacid sequence set forth as the antigenic site V.1 of a filovirus GP,such as any one of SEQ ID NOs: 21-25 or 59, wherein the peptide is nomore than 100 (such as no more than 75 or no more than 50) amino acidsin length and induces a neutralizing immune response to filovirus (suchas ebolavirus) in a subject.

In some embodiments, an isolated peptide is provided, wherein thepeptide comprises, consists essentially of, or consists of the aminoacid sequence set forth as the antigenic site V.10 of a filovirus GP,such as any one of SEQ ID NOs: 29-31 or 60, wherein the peptide is nomore than 100 (such as no more than 75 or no more than 50) amino acidsin length and induces a neutralizing immune response to filovirus (suchas ebolavirus) in a subject.

In some embodiments, an isolated peptide is provided, wherein thepeptide comprises, consists essentially of, or consists of the aminoacid sequence set forth as the antigenic site II.1 of a filovirus GP,such as any one of SEQ ID NOs: 47-50 or 61, wherein the peptide is nomore than 100 (such as no more than 75 or no more than 50) amino acidsin length and induces a neutralizing immune response to filovirus (suchas ebolavirus) in a subject.

In some embodiments, an isolated peptide is provided, wherein thepeptide comprises, consists essentially of, or consists of the aminoacid sequence set forth as the antigenic site IV.2 of a filovirus GP,such as any one of SEQ ID NOs: 51-52 or 62, wherein the peptide is nomore than 100 (such as no more than 75 or no more than 50) amino acidsin length and induces a neutralizing immune response to filovirus (suchas ebolavirus) in a subject.

Nucleic acid molecules encoding the disclosed immunogens and expressionvectors (such as an inactivated or attenuated viral vector) includingthe nucleic acid molecules are also provided.

Immunogenic compositions including the disclosed peptide (for example,linked to a carrier) or a nucleic acid molecule or vector encoding thepeptide are also provided. The immunogenic composition is suitable foradministration to a subject, and may also be contained in a unit dosageform. The immunogenic compositions can further include an adjuvant.

Methods of inducing an immune response to filovirus GP in a subject aredisclosed. In such methods a subject, such as a human subject, isadministered an effective amount of a disclosed immunogenic compositionto elicit the immune response. The subject can be, for example, a humansubject at risk of or having an ebolavirus infection.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Analysis of antibody repertoires elicited in adults afterfirst and second vaccinations with different doses of rVSVΔG-ZEBOV-GP.(FIG. 1A) Number of captured phage clones isolated using EBOV GFPDLaffinity selection with sera from adults after the first and secondvaccinations with 3 million, 20 million or 100 million PFUrVSVΔG-ZEBOV-GP vaccine (n=10 per group). (FIGS. 1B and 1C) Schematicalignments of the peptides recognized in sera after the first (FIG. 1B)and second (FIG. 1C) vaccinations, identified by panning with EBOV GFPDLAmino acid designations are based on the GP protein sequence encoded bythe complete EBOV GP gene (FIG. 9). SP, signal peptide; HR1, heptadrepeat 1; HR2, heptad repeat 2; TM, transmembrane domain; CT,cytoplasmic tail. Bars indicate identified inserts in GP sequence.Graphical distribution of representative clones with a frequency of ≥2obtained after affinity selection are shown. The horizontal positionsand the lengths of the bars indicate the peptide sequence displayed onthe selected phage clone to its homologous sequence in the EBOV GPsequence on alignment. The thickness of each bar represents thefrequency of repetitively isolated phage, with the scale shown below thealignment. The GFPDL affinity selection was performed in quadruplicate(two independent experiments by two different investigators, who wereblinded to sample identity).

FIGS. 2A-2C. Elucidation of antibody epitope profile against Ebola GPafter rVSVΔG-ZEBOV-GP vaccination in humans. (FIG. 2A) Antigenic siteswithin the EBOV GP recognized by serum antibodies after vaccination(based on data presented in FIG. 1) Amino acid designations are based onthe GP protein sequence encoded by the complete EBOV GP gene (FIG. 9).Epitopes previously described using MAbs are shown above the GPschematic. Critical residues for binding of MAbs in anti-EBOV cocktailsZMAb (1H3 (blue asterisks) and 2G4 and 4G7 (pink asterisks)), MB-003(13C6 (red asterisks), 6D8 and 13F6) and MAb KZ52 (Davidson, E. et al.J. Virol. 89, 10982-10992, 2015) are shown. (FIGS. 2B and 2C)Distribution and frequency of phage clones expressing each of the key GPantigenic sites after first (FIG. 2B) or second (FIG. 2C) vaccinationacross dosage groups (3 million PFU, 20 million PFU and 100 millionPFU). The number of clones encoding each antigenic site was divided bythe total number of EBOV GFPDL-selected clones for pooled sera from eachvaccine-dose group and represented as a percentage. Error bars represents.e.m. of 4 independent experiments. The GFPDL affinity selection wasperformed in quadruplicate.

FIG. 3A-3B. Structural representation of antigenic sites in EBOV GPidentified with GFPDL. Representations of individual antigenic sites onthe surface structures of a complete EBOV GP model (left) and solvedEBOV GP structure (PDB 3CSY) (Lee, J. E. et al. Nature 454, 177-182,2008) are shown (right) where available; antigenic sites in a monomer(chain A) are color coded as in FIG. 2. The EBOV GP structure used forcrystallography encompasses amino acid residues 33-189, 214-278, 299-310and 502-599 of the mature 676-aa GP sequence. The transmembrane (TM)domain is shown in orange, and the viral membrane is shown (gray bar) onthe model images. Sites I, II and VI on the model are shown in frontview. Sites II.1, 11.2, III, 111.1, IV, IV.1, IV.2, IV.3, V and V.1-V.7are shown in rear view. All sites (except site I) are depicted in frontview on the solved structure.

FIGS. 4A-4F. SPR-based analysis of sera from rVSVΔG-ZEBOV-GP-vaccinatedindividuals with EBOV GP purified protein. (FIGS. 4A and 4B) Totalbinding (RU) to purified mature GP from Mayinga (FIG. 4A) or Makona(FIG. 4B) EBOV strains in serum samples collected at different timepoints from adults receiving vaccine or placebo intramuscularly on day 0(DO) and day 28 (D28). Maximum RU values for GP binding by serumantibodies obtained from all individuals are shown. Data are mean±s.d.No significant differences were found between groups (P≥0.05,multiple-comparison adjustment using Bonferroni method). (FIG. 4C) EBOVneutralization endpoint titers of serum antibodies in pseudovirusneutralization assay (PsVNA) used for EBOV GFPDL-based epitope mapping.PsVNA80 titer refers to the highest serum dilutions required to achieve80% viral inhibition. (FIG. 4D) Correlation between maximum RU forpost-vaccination sera against Makona GP and homologous virusneutralization titers (PsVNA80; Spearman r=0.7598, P<0.0001) after thefirst (day 28) and second (day 56) vaccinations. (FIG. 4E) Polyclonalantibody affinity to EBOV GP after rVSVΔG-ZEBOV-GP vaccination. SPRanalysis of post-vaccination sera was performed with Makona GP todetermine the Kd of polyclonal serum antibodies from all individuals atdifferent post-vaccination time points. Horizontal bars indicate meanvalues. *P<0.05; **P<0.01; ***P<0.001. (FIG. 4F) Correlation ofGP-binding affinity, as measured by Kd of post-vaccination humanpolyclonal antibodies against Makona GP, with the homologous virusneutralization titers (PsVNA80; Spearman r=0.876, P<0.0001) after thefirst (day 28) and second (day 56) vaccinations. All SPR experimentswere performed twice.

FIGS. 5A-5F. Antibody isotypes in human serum binding to EBOV GP aftervaccination and the role of IgM antibodies in virus neutralization.(FIG. 5A) Isotypes of serum antibodies bound to EBOV GP for samplescollected from adults from each of the three vaccine-dose groups atdifferent time points, as measured in SPR. RU values for each anti-GPantibody isotype were divided by the total combined RU value for allisotypes for individual serum samples and represented as a percentage.(FIG. 5B) GP binding of IgG and IgM fractions purified from day 28(VSV-rGP-D28) sera of one of the three subjects with equivalentrepresentations of IgG and IgM isotypes in the EBOV GP-bound antibodies.(FIGS. 5C and 5D) Confirmation of the purity of isotype binding to GPusing antihuman IgG (FIG. 5C) and anti-human IgM (FIG. 5D) secondaryantibodies. (FIGS. 5E and 5F) Pseudovirus neutralization assay toevaluate purified IgG and IgM antibodies for virus neutralizationagainst Kikwit (FIG. 5E) and Makona (FIG. 5F) EBOV strains. The data arerepresented as the relative contributions of IgM and IgG antibodies tothe total neutralization observed for each sample. Error bars representmean±s.e.m. of 2 technical replicates. All SPR experiments werereplicated twice.

FIGS. 6A and 6B. Antigenic regions/sites of Zaire ebolavirus GP setforth as SEQ ID NO: 35 identified using GFPDL. The residues of SEQ IDNO: 35 for each sequence are indicated in the table. Table showingfrequency of antigenic sites for different vaccine dose groups afterfirst and second rVSVΔG-ZEBOV-GP vaccination. The frequency of the cloneper antigenic site is calculated by dividing the frequency of occurrenceof a particular clone by the total number of phage clones for eachvaccine dose.

FIG. 7. Sequence conservation of antigenic regions/sites among differentstrains and species of ebolavirus. The reference sequence used fordetermining sequence conservation was SEQ ID NO: 35, and the residuenumbering shown in the figure corresponds to SEQ ID NO: 35. Antigenicsites in the GP that are >70% conserved in diverse EBOV strains areshown in bold.

FIG. 8. Phage Titers from affinity selection of pooled sera withdifferent doses of rVSVΔG-ZEBOV-GP vaccine following first vaccinationusing Protein A/G, IgM and IgA matrices.

FIG. 9. Complete Zaire ebolavirus Mayinga (1976) GP gene translatedsequence (SEQ ID NO: 35) used for construction of Zaire ebolavirus-GFPDlibrary and depiction in FIGS. 1-3.

FIGS. 10A and 10B. Alignment of glycoprotein (GP) sequences from Zaireebolavirus Mayinga (1976, SEQ ID NO: 35), Kikwit (1995, SEQ ID NO: 36)and Makona (2014, SEQ ID NO: 37) strains.

FIG. 11. Random distribution of size and sequence of the EBOV-GFPDL.Sequencing of GP fragments expressed by the phages of the EBOV GFPDlibraries were aligned to the Zaire EBOV GP translated sequence (shownin FIG. 9).

FIG. 12. GFPDL based epitope mapping of protective MAbs 6D8 and 13F6used in the MB-003 cocktail. Graphical distribution of representativeclones with a frequency of >2, obtained after affinity selection, areshown. The common conserved minimal sequence (residues of SEQ ID NO: 35,residue numbering is shown in the figure) for each Mab identified usingGFPDL mapping is shown in the table compared to the sequence previouslyidentified ‘known site’ (Davidson, E. et al. J. Virol. 89, 10982-10992,2015). The reactivity of the GFPDL identified sequence to the respectiveMAb was confirmed by phage ELISA.

FIGS. 13A-13C. GFPDL based epitope mapping of cross-reactiveconformation dependent neutralizing and protective human MAb 289 and MAb324 from EBOV survivors (Flyak, A. I. et al. Cell 164, 392-405, 2016).The reactivity of the GFPDL identified sequence to the respective MAbwas confirmed by phage ELISA shown in FIGS. 13B and 13C using theindicated phage clones.

FIG. 14. Anti-GP reactivity of post-second vaccination sera for 20million and 100 million pfu vaccine dose in ELISA before and afterEBOVGFPDL adsorption. Post second vaccination sera from individualsvaccinated with 20 million and 100 million rVSVΔG-ZEBOV-GP vaccine dosewas adsorbed on EBOV GFPDL coated petri dishes. Binding to recombinantEBOV-GP is shown before and after GFPDL-adsorption (the after conditionis indicated by “M13 ads”) in ELISA using HRP conjugated goat anti-humanIgA IgG IgM specific antibody.

FIG. 15. Model of the complete Zaire Ebola GP monomer generated usingI-TASSER (left) showing transmembrane (TM) domain and membrane, andsolved crystal GP structure (PDB Id #3CSY; includes GP residues 33-189,214-278, 299-310 and 502-599) in monomeric form on right.

FIGS. 16A-16C. Anti-GP reactivity of post-first and post-secondvaccination sera for 20 million and 100 million pfu vaccine dose inELISA. The isotype of two-fold serial dilution of serum antibodies(starting at 200-fold serum dilution) bound to EBOV-GP are shown forserum samples collected at different time points from adults vaccinatedwith 20 million and 100 million rVSVΔG-ZEBOV-GP vaccine doseadministered groups as measured in ELISA using HRP conjugated anti-humanIgM muchain specific antibody (FIG. 16A), HRP conjugated anti-human IgGFc-chain specific antibody (FIG. 16B) and HRP conjugated anti-human IgAalpha-chain specific antibody (FIG. 16C).

FIG. 17. IgG subclass of human serum binding to EBOV-GP followingrVSVΔG-ZEBOV-GP vaccination. The subclass of total IgG of serumantibodies bound to EBOV-GP are shown for serum samples collected atdifferent time points from adults vaccinated with three differentrVSVΔG-ZEBOV-GP vaccine dose administered groups as measured in SPRexperiment. The resonance unit for each anti-GP antibody IgG subclasswas divided by the total resonance units for total bound IgG antibodiescombined for each sera and represented as a percentage.

FIGS. 18A-18C. Individual antibody repertoires elicited following firstvaccination with rVSVΔG-ZEBOV-GP vaccine using IgA, IgG and IgM specificcapture beads. (FIG. 18A) Number of captured phage clones isolated usingEBOV-GFPDL affinity selection with sera obtained from individualvaccinee after first vaccination with 20 or 100 million pfurVSVΔGZEBOV-GP vaccine dose group using IgA, IgG and IgM specificcapture beads by EBOVKikwit GP GFPDL. (FIG. 18B, FIG. 18C) Schematicalignment of the peptides recognized by IgG, IgM and IgA antibodiesfollowing first vaccination with 20 million (FIG. 18B) and 100 million(FIG. 18C) rVSVΔGZEBOV-GP vaccine, identified by panning with GFPDL. Theamino acid designation is based on the GP protein sequence encoded bythe complete EBOV GP gene (FIGS. 10A and 10B). Bars indicate identifiedinserts in GP sequence. The GP receptor binding region (RBR) and themucin like domain are indicated. Bars indicate identified inserts in GPsequence. Bar location indicates the homology of the displayed EBOV-GPprotein sequence on the phage clones after affinity selection. Graphicaldistribution of representative clone with a frequency of >2, obtainedafter affinity selection, are shown. The thickness of each barrepresents the frequency of repetitively isolated phage, with the scaleshown below the alignment.

FIGS. 19A-19C. Sequence alignment of GP proteins from Zaire ebolavirusMayinga (SEQ ID NO: 35), Zaire ebolavirus Kikwit (SEQ ID NO: 36), Zaireebolavirus Makona (SEQ ID NO: 37), Bundibugyo ebolavirus (SEQ ID NO:38), Sudan ebolavirus (SEQ ID NO: 39), Tai Forest ebolavirus (SEQ ID NO:41), and Marburg marburgvirus (SEQ ID NO: 42)

FIG. 20. Sequence of identified antigenic sites of Zaire ebolavirusMayinga GP (SEQ ID NO: 35).

FIG. 21. Rabbit immunization of GP antigenic site peptides induces highantibody binding to homologous and heterologous native GP.

FIG. 22. Conserved antigenic sites in GP1 induce neutralizingantibodies.

FIG. 23. Zaire ebolavirus challenge study in mice. Immunization withpeptides from Antigenic sites V.7 in GP1 and VI in GP2 inducessterilizing immunity, while other sites provide partial protectionagainst Ebola virus.

FIG. 24. Conserved antigenic sites in V.7 in GP1 and VI.1 in GP2 providesignificant protection from weight loss following Zaire ebolaviruschallenge, while other sites provide partial protection against Ebolavirus.

FIG. 25. Conserved antigenic sites in the C-terminal region of GP1 andGP2 generate strong binding antibodies to both Mayinga and Makona GP asmeasured by surface plasmon resonance.

FIG. 26. Sera from human Ebola virus survivors shows higher antibodybinding to diverse antigenic sites in GP than post-vaccination sera.

FIG. 27. Identification of GP antigenic sites that strongly correlatewith Ebola neutralization titers (PsVN).

FIG. 28. Spatial structure of linear neutralizing and protectiveantigenic sites in GP.

SEQUENCES

The nucleic and amino acid sequences listed herein are shown usingstandard letter abbreviations for nucleotide bases, and three lettercode for amino acids, as defined in 37 C.F.R. 1.822. Only one strand ofeach nucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand. TheSequence Listing is submitted as an ASCII text file in the form of thefile named “Sequence.txt” (˜28 kb), which was created on Oct. 30, 2017which is incorporated by reference herein.

SEQ ID NOs: 1-34 and 43-61 are the amino acid sequences of immunogenicpeptides, as follows:

SEQ ID NO: 1:  TTEDHKIMASENSSAMVQVHSQGREAAVSH SEQ ID NO: 8: TX₁₀EDHKIMASENSSAMVQVHSQGRX₁₁AAVSHwherein: X₁₀ is T or N; and X₁₁ is E or K.Antigenic site II.1 peptides, residues 152-220: SEQ ID NO: 47: AFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLR EPVNATEDPSSGYYSTTIRY (Zaire ebolavirus Mayinga, Kikwit, Makona) SEQ ID NO: 48: AFHKEGAFFLYDRLASTIIYRSTTFSEGVVAFLILPKTKKDFFQSPPLHEPANMTTDPSSYYHTVTLNY (Bundibugyo ebolavirus) SEQ ID NO: 49: AFHKDGAFFLYDRLASTVIYRGVNFAEGVIAFLILAKPKETFLQSPPIRE AVNYTENTSSYYATSYLEY (Sudan ebolavirus) SEQ ID NO: 50: AFHKEGAFFLYDRLASTIIYRGTTFAEGVIAFLILPKARKDFFQSPPLHEP ANMTTDPSSYYHTTTINY (Ta ï  Forest ebolavirus) SEQ ID NO: 61: ALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQG QGYRHMNLTSTNKYWTSSNGT (Margburg margburgvirus) Antigenic site IV.1 peptides, residues 282-305:SEQ ID NO: 17:  DTTIGEWAFWETKKNLTRKIRSEE,Zaire ebolavirus Mayinga, Kikwit, Makona) SEQ ID NO: 18: DTGVGEWAFVVENKKNFTKTLSSEE (Bundibugyo ebolavirus) SEQ ID NO: 19: NADIGEWAFVVENKKNLSEQLRGEE (Sudan ebolavirus) SEQ ID NO: 20: DTSMGEWAFVVENKKNFKKTLSSEE (Ta ï  Forest ebolavirus) SEQ ID NO: 58: DEDLATSGSGSGEREPHTTSD (Margburg margburgvirus)Antigenic site IV.2 peptides, residues 286-296: SEQ ID NO: 51: GEWAFVVETKKN (Zaire ebolavirus Mayinga, Kikwit, Makona); SEQ ID NO: 52: GEWAFVVENKKN (Bundibugyo ebolavirus, Sudan ebolavirus, Ta ï Forest ebolavirus); SEQ ID NO: 62:  ATSGSGSGER (Margburg margburgvirus) Antigenic site  V.1 peptides, residues 343-368:SEQ ID NO: 21:  ASENSSAMVQVHSQGREAAVSHLTTL(Zaire ebolavirus Mayinga, Kikwit) SEQ ID NO: 22: ASENSSAMVQVHSQGRKAAVSHLTTL (Zaire ebolavirus Makona) SEQ ID NO: 23: VPKDPASVVQVRDLQRENTVPTSP (Bundibugyo ebolavirus) SEQ ID NO: 24: VPKNSPGVVPLHIPEGETTLPSQNST (Sudan ebolavirus) SEQ ID NO: 25: VSEDSTPVVQMQNIKGKDTMPTTV (Ta ï  Forest ebolavirus) SEQ ID NO: 59: LDKNNTTAQPSMPPHNTTTISTNNTS (Margburg margburgvirus)Antigenic site V.6 peptides, residues 457-484 and 546-484:SEQ ID NO: 2:  ETAGNNNTHHQDTGEESASSGKLGLITN(Zaire ebolavirus Mayinga, Kikwit, Makona) SEQ ID NO: 26: MITSHDTDSNRPNPIDISESTEPGLLTN (Bundibugyo ebolavirus) SEQ ID NO: 27: LTTPENITTAVKTVLPQESTSNGLITS (Sudan ebolavirus) SEQ ID NO: 28: LPEQHTAASAIPRAVHPDELSGPGFLTN (Ta ï  Forest ebolavirus) SEQ ID NO: 56: LWREGDMFPFLDGLINAPIDFDPVPTK (Margburg margburgvirus) SEQ ID NO: 43: SETAGNNNTHHQDTGEESASSGKLGLITN (Zaire ebolavirus Mayinga, Kikwit, Makona)SEQ ID NO: 44:  TMITSHDTDSNRPNPIDISESTEPGLLTN (Bundibugyo ebolavirus)SEQ ID NO: 45:  TLTTPENITTAVKTVLPQESTSNGLITS (Sudan ebolavirus)SEQ ID NO: 46:  TLPEQHTAASAIPRAVHPDELSGPGFLTN (Ta ï  Forest ebolavirus)SEQ ID NO: 57:  ILWREGDMFPFLDGLINAPIDFDPVPTK (Margburg margburgvirus)Antigenic site V.7 peptides, residues 469-498: SEQ ID NO: 3: TGEESASSGKLGLITNTIAGVAGLITGGRR(Zaire ebolavirus Mayinga, Kikwit, Makona) SEQ ID NO: 32: NPIDISESTEPGLLTNTIRGVANLLTGSRR (Bundibugyo ebolavirus) SEQ ID NO: 33: TVLPQESTSNGLITSTVTGILGSLGLRKR (Sudan ebolavirus) SEQ ID NO: 34: RAVHPDELSGPGFLTNTIRGVTNLLTGSRR (Ta ï  forest ebolavirus) SEQ ID NO: 55: GLINAPIDFDPVPNTKTIFDESSSSGASAE (Margburg margburgvirus)Antigenic site V.10 peptides, residues 520-547: SEQ ID NO: 29: TQDEGAAIGLAWIPYFGPAAEGIYIEGL (Zaire ebolavirus Mayinga) SEQ ID NO: 30: TQDEGAAIGLAWIPYFGPAAEGIYTEGL(Zaire ebolavirus Kikwit, Makona, Sudan ebolavirus) SEQ ID NO: 31: TQDEGAAIGLAWIPYFGPAAEGIYTEGI (Bundibugyo ebolavirus, Ta ï Forest ebolavirus) SEQ ID NO: 60:  VQEDDLAAGLSWIPFFGPGIEGLYTAGL(Margburg margburgvirus)Antigenic site VI peptides, residues 617-645 or 617-646: SEQ ID NO: 9 (KNITDKIX₁QIIHDFX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWW, wherein X₁ is D or N,X₂ is V or I, X₃ is K or N, X₄ is T, P, or N, X₅ is D or N, X₆ is G, T, D, or N, X₇ is D or N, X₈ is N, D, orG, and X₉ is D or S (Antigenic site VI consensus,) SEQ ID NO: 10: KNITDKIDQIIHDFVDKTLPDQGDNDNWW (Zaire ebolavirus Mayinga, Kikwit, Makona)SEQ ID NO: 11:  KNITDKINQIIHDFIDKPLPDQTDNDNWW (Bundibugyo ebolavirus)SEQ ID NO: 12:  KNITDKIDQIIHDFIDNPLPNQDNDDNWW (Sudan ebolavirus)SEQ ID NO: 13:  KNITDKINQIIHDFVDNNLPNQNDGSNWW (Ta ï  Forest ebolavirus)SEQ ID NO: 53: KNISEQIDQIKKDEQKEGTGWGLGGKWW (Margburg margburgvirus)SEQ ID NO: 7:  KNITDKIX₁QIIHDFX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWWT,wherein: X₁ is D or N, X₂ is V and I, X₃ is K or N, X₄ is T, P, or N, X₅ is D or N, X₆ is G, T, D, or N, X₇ is D or N, X₈ is N,D, or G; and X₉ is D or S SEQ ID NO: 4:  KNITDKIDQIIHDFVDKTLPDQGDNDNWWT(Zaire ebolavirus Mayinga, Kikwit, Makona) SEQ ID NO: 14: KNITDKINQIIHDFIDKPLPDQTDNDNWWT (Bundibugyo ebolavirus) SEQ ID NO: 15:  KNITDKIDQIIHDFIDNPLPNQDNDDNWWT  (Sudan ebolavirus) SEQ ID NO: 16: KNITDKINQIIHDFVDNNLPNQNDGSNWWT  (Ta ï  Forest ebolavirus)SEQ ID NO: 54:  KNISEQIDQIKKDEQKEGTGWGLGGKWWT  (Margburg margburgvirus)Antigenic site VI.1 peptides, residues 630-646: SEQ ID NO: 6 (FX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWWT, wherein: X₂ is selected from V and I; X₃from K and N; X₄ from T, P, and N; X₅ and X₇ from D and N;X₆ from G, T, D, and N; X₈ from N, D, and G; and X₉ from D and SSEQ ID NO: 5:  FVDKTLPDQGDNDNWWT (Zaire ebolavirus Mayinga, Kikwit, Makona)Residues 14-30 of SEQ ID NO: 14:  KNITDKINQIIHDFIDKPLPDQTDNDNWWT(Bundibugyo ebolavirus) Residues 14-30 of SEQ ID NO: 15: KNITDKIDQIIHDFIDNPLPNQDNDDNWWT  (Sudan ebolavirus)Residues 14-30 of SEQ ID NO: 16:  KNITDKINQIIHDFVDNNLPNQNDGSNVVWT  (Ta ï Forest ebolavirus)SEQ ID NO: 35 is an exemplary amino acid sequence of a precursor of the GP from theMayinga Strain of Zaire ebolavirus (GENBANK Acc. No. AIO11753.1, which is incorporated byreference herein in its entirety).MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVFSEQ ID NO: 36 is an exemplary amino acid sequence of a precursor of the GP from the KikwitStrain of Zaire ebolavirus (GENBANK Acc. No. AIO11753.1, which is incorporated by reference hereinin its entirety).MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTAVSNRAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQPPTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPPATTAAGPLKAENTNTSKGTDLLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRARREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYTEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVFSEQ ID NO: 37 is an exemplary amino acid sequence of a precursor of the GP from the MakonaStrain of Zaire ebolavirus (GENBANK Acc. No. AIO11753.1, which is incorporated by reference hereinin its entirety).MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSTNQLRSVGLNLEGNGVATSIPLGVIHNSTLQVSDVDKLVCRDKLSDVPSVTKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSGYYSTTIRYQATGFGTNETESHPLREPVNATEDPSYLFEVDNLTYVQLESRFTPQFLLQLNETIYASGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTAVSNGPKNISGQSPARTSSDPETNTTNEDHKIMASENSSAMVQVHSQGRKAAVSHLTTLATISTSPQPPTTKTGPDNSTHNTPVYKLDISEATQVGQHHRRADNDSTASDTPPATTAAGPLKAENTNTSKSADSLDLATTTSPQNYSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREVIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYTEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNI TDKI DQII HDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVFSEQ ID NO: 38 is an exemplary amino acid sequence of a precursor of the GP from Bundibugyoebolavirus (GENBANK Acc. No. AGL73460.1, which is incorporated by reference herein in its entirety).MVTSGILQLPRERFRKTSFFVWVIILFHKVFPIPLGVVHNNTLQVSDIDKLVCRDKLSSTSQLKSVGLNLEGNGVATDVPTATKRWGFRAGVPPKVVNYEAGEWAENCYNLDIKKADGSECLPEAPEGVRGFPRCRYVHKVSGTGPCPEGFAFHKEGAFFLYDRLASTIIYRSTTFSEGVVAFLILPKTKKDFFQSPPLHEPANMTTDPSSYYHTVTLNYVADNFGTNMTNFLFQVDHLTYVQLEPRFTPQFLVQLNETIYTNGRRSNTTGTLIWKVNPTVDTGVGEWAFWENKKNFTKTLSSEELSVILVPRAQDPGSNQKTKVTPTSFANNQTSKNHEDLVPKDPASVVQVRDLQRENTVPTSPLNTVPTTLIPDTMEEQTTSHYELPNISGNHQERNNTAHPETLANNPPDNTTPSTPPQDGERTSSHTTPSPRPVPTSTIHPTTRETQIPTTMITSHDTDSNRPNPIDISESTEPGLLTNTIRGVANLLTGSRRTRREITLRTQAKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYTEGIMHNQNGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFIDKPLPDQTDNDNWWTGWRQWVPAGIGITGVIIAVIALLCICKFLLSEQ ID NO: 39 is an exemplary amino acid sequence of a precursor of the GP from Sudanebolavirus (GENBANK Acc. No. ACR33190.1, which is incorporated by reference herein in its entirety).MEGLSLLQLPRDKFRKSSFFVWVIILFQKAFSMPLGVVTNSTLEVTEIDQLVCKDHLASTDQLKSVGLNLEGSGVSTDIPSATKRWGFRSGVPPKVFSYEAGEWAENCYNLEIKKPDGSECLPPPPDGVRGFPRCRYVHKAQGTGPCPGDYAFHKDGAFFLYDRLASTVIYRGVNFAEGVIAFLILAKPKETFLQSPPIREAVNYTENTSSYYATSYLEYEIENFGAQHSTTLFKINNNTFVLLDRPHTPQFLFQLNDTIHLHQQLSNTTGKLIWTLSEQLRGEELSFDANINADIGEWAFWENKKNLETLSLNETEDDDATSSRTTKGRISDRATRKYSDLVPKDSPGMVSLHVPEGETTLPSQNSTEGRRVDVNTQETITETTATIIGTNGNNMQISTIGTGLSSSQILSSSPTMAPSPETQTSTTYTPKLPVMTTEESTTPPRNSPGSTTEAPTLTTPENITTAVKTVLPQESTSNGLITSTVTGILGSLGLRKRSRRQVNTRATGKCNPNLHYWTAQEQHNAAGIAWIPYFGPGAEGIYTEGLMHNQNALVCGLRQLANETTQALQLFLRATTELRTYTILNRKAIDFLLRRWGGTCRILGPDCCIEPHDWTKNITDKINQIIHDFIDNPLPNQDNDDNWWTGWRQWIPAGIGITGIIIAIIALLCVCKLLCSEQ ID NO: 40 is an exemplary amino acid sequence of a precursor of the GP from Restonebolavirus (GENBANK Acc. No. AAC54891.1, which is incorporated by reference herein in itsentirety).MGSGYQLLQLPRERFRKTSFLVWVIILFQRAISMPLGIVTNSTLKATEIDQLVCRDKLSSTSQLKSVGLNLEGNGIATDVPSATKRWGFRSGVPPKVVSYEAGEWAENCYNLEIKKSDGSECLPLPPDGVRGFPRCRYVHKVQGTGPCPGDLAFHKNGAFFLYDRLASTVIYRGTTFTEGVVAFLILSEPKKHFWKATPAHEPVNTTDDSTSYYMTLTLSYEMSNFGGKESNTLFKVDNHTYVQLDRPHTPQFLVQLNETLRRNNRLSNSTGRLTWTLDPKIEPDVGEWAFWETKKNFSQQLHGENLHFQILSTHTNNSSDQSPAGTVQGKISYHPPTNNSELVPTDSPPVVSVLTAGRTEEMSTQGLTNGETITGFTANPMTTTIAPSPTMTSEVDNNVPSEQPNNTASIEDSPPSASNETIDHSEMNPIQGSNNSAQSPQTKTTPAPTASPMTQDPQETANSSKLGTSPGSAAEPSQPGFTINTVSKVADSLSPTRKQKRSVRQNTANKCNPDLHYWTAVDEGAAVGLAWIPYFGPAAEGIYIEGVMHNQNGLICGLRQLANETTQALQLFLRATTELRTYSLLNRKAIDFLLQRWGGTCRILGPSCCIEPHDWTKNITDEINQIKHDFIDNPLPDHGDDLNLWTGWRQWIPAGIGIIGVIIAIIALLCICKILCSEQ ID NO: 41 is an exemplary amino acid sequence of a precursor of the GP from Taï  Forestebolavirus (GENBANK Acc. No. ACI28632.1, which is incorporated by reference herein in its entirety).MGASGILQLPRERFRKTSFFVWVIILFHKVFSIPLGVVHNNTLQVSDIDKFVCRDKLSSTSQLKSVGLNLEGNGVATDVPTATKRWGFRAGVPPKVVNCEAGEWAENCYNLAIKKVDGSECLPEAPEGVRDFPRCRYVHKVSGTGPCPGGLAFHKEGAFFLYDRLASTIIYRGTTFAEGVIAFLILPKARKDFFQSPPLHEPANMTTDPSSYYHTTTINYVVDNFGTNTTEFLFQVDHLTYVQLEARFTPQFLVLLNETIYSDNRRSNTTGKLIWKINPTVDTSMGEWAFWENKKNFTKTLSSEELSFVPVPETQNQVLDTTATVSPPISAHNHAAEDHKELVSEDSTPVVQMQNIKGKDTMPTTVTGVPTTTPSPFPINARNTDHTKSFIGLEGPQEDHSTTQPAKTTSQPTNSTESTTLNPTSEPSSRGTGPSSPTVPNTTESHAELGKTTPTTLPEQHTAASAIPRAVHPDELSGPGFLTNTIRGVTNLLTGSRRKRRDVTPNTQPKCNPNLHYWTALDEGAAIGLAWIPYFGPAAEGIYTEGIMENQNGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPQDWTKNITDKIDQIIHDFVDNNLPNQNDGSNWWTGWKQWVPAGIGITGVIIAIIALLCICKFMLSEQ ID NO: 42 is an exemplary amino acid sequence of a precursor of the GP from Marburgmargburgvirus (GENBANK Acc. No. AAR85456.1, which is incorporated by reference herein in itsentirety).MKTTCFLISLILIQGTKNLPILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTARPEIKLTSTPTDATKLNTTDPSSDOEDLATSGSGSGEREPHTTSDAVTKQGLSSTMPPTPSPQPSTPQQGGNNTNHSQDAVTELDKNNTTAQPSMPPHNTTTISTNNTSKHNFSTLSAPLQNTTNDNTQSTITENEQTSAPSITTLPPTGNPTTAKSTSSKKGPATTAPNTTNEHFTSPPPTPSSTAQHLVYFRRKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG

DETAILED DESCRIPTION I. Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishers, 2009; and Meyers et al. (eds.), The Encyclopedia of CellBiology and Molecular Medicine, published by Wiley-VCH in 16 volumes,2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” It isfurther to be understood that any and all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescriptive purposes, unless otherwise indicated. Although many methodsand materials similar or equivalent to those described herein can beused, particular suitable methods and materials are described herein. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting. To facilitatereview of the various embodiments, the following explanations of termsare provided:

Adjuvant: A vehicle used to enhance antigenicity. In some embodiments,an adjuvant can include a suspension of minerals (alum, aluminumhydroxide, or phosphate) on which antigen is adsorbed; or water-in-oilemulsion, for example, in which antigen solution is emulsified inmineral oil (Freund incomplete adjuvant), sometimes with the inclusionof killed mycobacteria (Freund's complete adjuvant) to further enhanceantigenicity (inhibits degradation of antigen and/or causes influx ofmacrophages). In some embodiments, the adjuvant used in a disclosedimmunogenic composition is a combination of lecithin and carbomerhomopolymer (such as the ADJUPLEX™ adjuvant available from AdvancedBioAdjuvants, LLC, see also Wegmann, Clin Vaccine Immunol, 22(9):1004-1012, 2015). Additional adjuvants for use in the disclosedimmunogenic compositions include the QS21 purified plant extract, MatrixM, AS like adjuvants including AS01, MF59, ALFQ or other oil in water orwater in oil adjuvants. Immunostimulatory oligonucleotides (such asthose including a CpG motif) can also be used as adjuvants. Adjuvantsinclude biological molecules (a “biological adjuvant”), such ascostimulatory molecules. Exemplary adjuvants include IL-2, RANTES,GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL andtoll-like receptor (TLR) agonists, such as TLR-9 agonists. Additionaldescription of adjuvants can be found, for example, in Singh (ed.)Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007).Adjuvants can be used in combination with the disclosed immunogens.

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intravenous, the composition (such as a compositionincluding a disclosed immunogen) is administered by introducing thecomposition into a vein of the subject. Exemplary routes ofadministration include, but are not limited to, oral, injection (such assubcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), sublingual, rectal, transdermal (for example, topical),intranasal, vaginal, and inhalation routes.

Antibody: An immunoglobulin, antigen-binding fragment, or derivativethereof, that specifically binds and recognizes an analyte (antigen),such as a peptide from ebolavirus GP. The term “antibody” is used hereinin the broadest sense and encompasses various antibody structures,including but not limited to monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments, so long as they exhibit the desired antigen-bindingactivity. Non-limiting examples of antibodies include, for example,intact immunoglobulins and variants and fragments thereof that retainbinding affinity for the antigen. Examples of antibody fragments includebut are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies;linear antibodies; single-chain antibody molecules (e.g. scFv); andmultispecific antibodies formed from antibody fragments. Antibodyfragments include antigen binding fragments either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed),Antibody Engineering, Vols. 1-2, 2^(nd) Ed., Springer Press, 2010).Light and heavy chain variable regions contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs” (see, e.g., Kabat etal., Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991). The framework region of an antibody,that is the combined framework regions of the constituent light andheavy chains, serves to position and align the CDRs in three-dimensionalspace. The CDRs are primarily responsible for binding to an epitope ofan antigen.

Carrier: An immunogenic molecule to which a peptide can be linked toenhance an immune response to the peptide. Carriers are chosen toincrease the immunogenicity of the antigen and/or to elicit antibodiesagainst the carrier which are diagnostically, analytically, and/ortherapeutically beneficial. Useful carriers include polymeric carriers,which can be natural (for example, proteins from bacteria or viruses),semi-synthetic or synthetic materials containing one or more functionalgroups to which a reactant moiety can be attached.

Conjugate: A complex of at least two heterologous molecules linkedtogether. In a non-limiting example, an ebolavirus peptide as disclosedherein is conjugated to a protein carrier by a linker.

Consists essentially of and Consists Of: A polypeptide comprising anamino acid sequence that consists essentially of a specified amino acidsequence does not include any additional amino acid residues. However,the residues in the polypeptide can be modified to include non-peptidecomponents, such as labels (for example, fluorescent, radioactive, orsolid particle labels), sugars or lipids, and the N- or C-terminus ofthe polypeptide can be joined (for example, by peptide bond) toheterologous amino acids, such as a cysteine (or other) residue in thecontext of a linker for conjugation chemistry. A polypeptide thatconsists of a specified amino acid sequence does not include anyadditional amino acid residues, nor does it include additionalbiological components, such as nucleic acids lipids, sugars, nor does itinclude labels. However, the N- or C-terminus of the polypeptide can bejoined (for example, by peptide bond) to heterologous amino acids, suchas a peptide tag, or a cysteine (or other) residue in the context of alinker for conjugation chemistry.

A polypeptide that consists or consists essentially of a specified aminoacid sequence can be glycosylated or have an amide modification. Apolypeptide that consists of or consists essentially of a particularamino acid sequence can be linked via its N- or C-terminus to aheterologous polypeptide, such as in the case of a fusion proteincontaining a first polypeptide consisting or a first sequence that islinked (via peptide bond) to a heterologous polypeptide consisting of asecond sequence. In another example, the N- or C-terminus of apolypeptide that consists of or consists essentially of a particularamino acid sequence can be linked to a peptide linker (via peptide bond)that is further linked to one or more additional heterologouspolypeptides. In a further example, the N- or C-terminus of apolypeptide that consists of or consists essentially of a particularamino acid sequence can be linked to one or more amino acid residuesthat facilitate further modification or manipulation of the polypeptide.

Control: A reference standard. In some embodiments, the control is anegative control, such as sample obtained from a healthy patient notinfected with EBOV. In other embodiments, the control is a positivecontrol, such as a tissue sample obtained from a patient diagnosed withEBOV infection. In still other embodiments, the control is a historicalcontrol or standard reference value or range of values (such as apreviously tested control sample, such as a group of EBOV patients withknown prognosis or outcome, or group of samples that represent baselineor normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, or at least about 500%.

Ebolavirus: A genus of enveloped, non-segmented, negative-sense,single-stranded RNA viruses that cause ebolavirus disease (EVD),formerly known as Ebola hemorrhagic fever (EHF), in humans. Ebolavirusesspread through human-to-human transmission, with infection resultingfrom direct contact with blood, secretions, organs or other bodilyfluids of infected people, and indirect contact with environmentscontaminated by such fluids. These may include other Filoviruses.

The symptoms of ebolavirus infection and EVD are well-known. Briefly, inhumans, ebolavirus has an initial incubation period of 2 to 21 days (7days on average, depending on the species) followed by a rapid onset ofnon-specific symptoms such as fever, extreme fatigue, gastrointestinalcomplaints, abdominal pain, anorexia, headache, myalgias and/orarthralgias. These initial symptoms last for about 2 to 7 days afterwhich more severe symptoms related to hemorrhagic fever occur, includinghemorrhagic rash, epistaxis, mucosal bleeding, hematuria, hemoptysis,hematemesis, melena, conjunctival hemorrhage, tachypnea, confusion,somnolence, and hearing loss. In general, the symptoms last for about 7to14 days after which recovery may occur. Death can occur 6 to 16 daysafter the onset of symptoms. People are infectious as long as theirblood and secretions contain the virus, which in some instances can bemore than 60 days.

Immunoglobulin M (IgM) antibodies to the virus appear 2 to 9 days afterinfection whereas immunoglobulin G (IgG) antibodies appear approximately17 to 25 days after infection, which coincides with the recovery phase.In survivors of EVD, both humoral and cellular immunity are detected,however, their relative contribution to protection is unknown.

Five distinct species of Ebolavirus are known, including Bundibugyoebolavirus, Reston ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus,and Zaire ebolavirus. Bundibugyo ebolavirus, Sudan ebolavirus, and Zaireebolavirus have been associated with large outbreaks of EVD in Africaand reported case fatality rates of up to 90%. Exemplary amino acidsequences of GP from Bundibugyo ebolavirus, Reston ebolavirus, Sudanebolavirus, Taï Forest ebolavirus, and Zaire ebolavirus are set forth asSEQ ID NOs: 25-29.

The ebolavirus genome includes about 19 kb, which encode sevenstructural proteins including NP (a nucleoprotein), VP35 (a polymerasecofactor), VP30 (a transcriptional activator), VP24, L (a RNApolymerase), and GP (a glycoprotein).

Ebolavirus glycoprotein (GP): The virion-associated transmembraneglycoprotein of Ebolavirus is initially synthesized as a precursorprotein of about 676 amino acids in size, designated GP₀. Individual GP₀polypeptides form a homotrimer and undergo glycosylation as well asprocessing to remove the signal peptide, and cleavage by a cellularprotease between approximately positions 501/502 to generate separateGP₁ and GP₂ polypeptide chains, which remain associated via disulfidebonds as GP₁/GP₂ protomers within the homotrimer. The extracellular GP₁trimer (approx. 140 kDa) is derived from the amino-terminal portion ofthe GP₀ precursors, and the GP₂ trimer (approx. 26 kDa), which includesextracellular, transmembrane, and cytosolic domains, is derived from thecarboxyl-terminal portion of the GP₀ precursors. GP₁ is responsible forattachment to new host cells while GP₂ mediates fusion with those cells.

Comparisons of the predicted amino acid sequences for the GPs of thedifferent ebolaviruses show conservation of amino acids in theamino-terminal and carboxy-terminal regions with a highly variableregion in the middle of the protein (Feldmann el al., Virus Res. 24:1-19, 1992). The GPs of the ebolaviruses are highly glycosylated andcontain both N-linked and O-linked carbohydrates that contribute up to50% of the molecular weight of the protein. Most of the glycosylationsites are found in the central variable region of GP.

The numbering used in the disclosed Filovirus GPs and fragments thereofis relative to the Zaire ebolavirus GP protein set forth as SEQ ID NO:35, unless context indicates otherwise.

Effective amount: An amount of agent, such as an immunogen, that issufficient to generate a desired response, such as an immune response ina subject. It is understood that to obtain a protective immune responseagainst an antigen of interest can require multiple administrations of adisclosed immunogen, and/or administration of a disclosed immunogen asthe “prime” in a prime boost protocol wherein the boost immunogen can bedifferent from the prime immunogen. Accordingly, an effective amount ofa disclosed immunogen can be the amount of the immunogen sufficient toelicit a priming immune response in a subject that can be subsequentlyboosted with the same or a different immunogen to generate a protectiveimmune response.

In one example, a desired response is to induce an immune response thatinhibits or prevents Ebola virus infection in a subject. For example,administration of an effective amount of a disclosed ebolavirus GPpeptide can induce an immune response in a subject that inhibitssubsequent infection of the subject by an ebolavirus.

Expression: Transcription or translation of a nucleic acid sequence. Forexample, a gene is expressed when its DNA is transcribed into an RNA orRNA fragment, which in some examples is processed to become mRNA. A genemay also be expressed when its mRNA is translated into an amino acidsequence, such as a protein or a protein fragment. In a particularexample, a heterologous gene is expressed when it is transcribed into anRNA. In another example, a heterologous gene is expressed when its RNAis translated into an amino acid sequence. The term “expression” is usedherein to denote either transcription or translation. Regulation ofexpression can include controls on transcription, translation, RNAtransport and processing, degradation of intermediary molecules such asmRNA, or through activation, inactivation, compartmentalization ordegradation of specific protein molecules after they are produced.

Expression control sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (ATG) in front of a protein-encoding gene, splicing signals forintrons, maintenance of the correct reading frame of that gene to permitproper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

A polynucleotide can be inserted into an expression vector that containsa promoter sequence which facilitates the efficient transcription of theinserted genetic sequence of the host. The expression vector typicallycontains an origin of replication, a promoter, as well as specificnucleic acid sequences that allow phenotypic selection of thetransformed cells.

Expression vector: A vector comprising a recombinant polynucleotidecomprising expression control sequences operatively linked to anucleotide sequence to be expressed. An expression vector comprisessufficient cis-acting elements for expression; other elements forexpression can be supplied by the host cell or in an in vitro expressionsystem. Expression vectors include cosmids, plasmids (e.g., naked orcontained in liposomes) and viruses (e.g., lentiviruses, retroviruses,adenoviruses, and adeno-associated viruses) that incorporate therecombinant polynucleotide.

Heterologous: Originating from a different genetic source. A nucleicacid molecule that is heterologous to a cell originated from a geneticsource other than the cell in which it is expressed. Methods forintroducing a heterologous nucleic acid molecule in a cell or organisminclude, for example, transformation with a nucleic acid, includingelectroporation, lipofection, particle gun acceleration, and homologousrecombination.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies. “Priming an immune response” refers to treatment of asubject with a “prime” immunogen to induce an immune response that issubsequently “boosted” with a boost immunogen. Together, the prime andboost immunizations produce the desired immune response in the subject.“Enhancing an immune response” refers to co-administration of anadjuvant and an immunogenic agent, wherein the adjuvant increases thedesired immune response to the immunogenic agent compared toadministration of the immunogenic agent to the subject in the absence ofthe adjuvant.

Immunogen: A protein or a portion thereof that is capable of inducing animmune response in a mammal, such as a mammal infected or at risk ofinfection with a pathogen.

Immunogenic composition: A composition comprising a disclosed immunogen,or a nucleic acid molecule or vector encoding a disclosed immunogen,that elicits a measurable CTL response against the immunogen, or elicitsa measurable B cell response (such as production of antibodies) againstthe immunogen, when administered to a subject. It further refers toisolated nucleic acids encoding an immunogen, such as a nucleic acidthat can be used to express the immunogen (and thus be used to elicit animmune response against this immunogen). For in vivo use, theimmunogenic composition will typically include the protein or nucleicacid molecule in a pharmaceutically acceptable carrier and may alsoinclude other agents, such as an adjuvant.

Inhibiting a disease or condition: Reducing the full development of adisease or condition in a subject, for example, reducing the fulldevelopment of Ebola virus disease in a subject who has an Ebola virusinfection, and/or reducing Ebola virus infection in a subject orpopulation of subjects at risk thereof. This includes neutralizing,antagonizing, prohibiting, preventing, restraining, slowing, disrupting,stopping, or reversing progression or severity of the disease orcondition.

Inhibiting a disease or condition refers to a prophylactic interventionadministered before the disease or condition has begun to develop (forexample, by vaccinating a subject at risk of EBOV infection, but notinfected by EBOV, with an ebolavirus GP peptide immunogen as disclosedherein) that reduces subsequent development of the disease or condition,and also to amelioration of one or more signs or symptoms of the diseaseor condition following development. The term “ameliorating,” withreference to inhibiting a disease or condition refers to any observablebeneficial effect of the intervention intended to inhibit the disease orcondition. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the disease or condition in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease or condition, a slower progression of thedisease or condition, an improvement in the overall health or well-beingof the subject, a reduction in infection, or by other parameters wellknown in the art that are specific to the particular disease orcondition.

In some embodiments, an immune response induced by administering aneffective amount of an ebolavirus GP peptide immunogen as disclosedherein inhibits infection of a human subject by ebolaviruses, forexample, by at least 50% (such as at least 60%, at least 70%, at least80%, at least 90%, or more) compared to a suitable control.

Isolated: An “isolated” biological component has been substantiallyseparated or purified away from other biological components, such asother biological components in which the component naturally occurs,such as other chromosomal and extrachromosomal DNA, RNA, and proteins.Proteins, peptides, nucleic acids, and viruses that have been “isolated”include those purified by standard purification methods. Isolated doesnot require absolute purity, and can include protein, peptide, nucleicacid, or virus molecules that are at least 50% isolated, such as atleast 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

Linked: The term “linked” means joined together, either directly orindirectly. For example, a first moiety may be covalently ornoncovalently (e.g., electrostatically) linked to a second moiety. Thisincludes, but is not limited to, covalently bonding one molecule toanother molecule, noncovalently bonding one molecule to another (e.g.electrostatically bonding), non-covalently bonding one molecule toanother molecule by hydrogen bonding, non-covalently bonding onemolecule to another molecule by van der Waals forces, and any and allcombinations of such couplings. Indirect attachment is possible, such asby using a “linker”. In several embodiments, linked components areassociated in a chemical or physical manner so that the components arenot freely dispersible from one another, at least until contacting orentering a cell, such as an immune cell.

Linker: One or more molecules or groups of atoms positioned between twomoieties. Typically, linkers are bifunctional, i.e., the linker includesa functional group at each end, wherein the functional groups are usedto couple the linker to the two moieties. The two functional groups maybe the same, i.e., a homobifunctional linker, or different, i.e., aheterobifunctional linker. In several embodiments, a peptide linker canbe used to link the C-terminus of a first protein to the N-terminus of asecond protein. Non-limiting examples of peptide linkers includeglycine-serine peptide linkers, which are typically not more than 10amino acids in length. Typically, such linkage is accomplished usingmolecular biology techniques to genetically manipulate DNA encoding thefirst polypeptide linked to the second polypeptide by the peptidelinker.

Nucleic acid molecule: A polymeric form of nucleotides, which mayinclude both sense and anti-sense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers of the above. A nucleotide refers toa ribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. The term “nucleic acid molecule” as used herein issynonymous with “nucleic acid” and “polynucleotide.” A nucleic acidmolecule is usually at least 10 bases in length, unless otherwisespecified. The term includes single- and double-stranded forms of DNA. Apolynucleotide may include either or both naturally occurring andmodified nucleotides linked together by naturally occurring and/ornon-naturally occurring nucleotide linkages. “cDNA” refers to a DNA thatis complementary or identical to an mRNA, in either single stranded ordouble stranded form. “Encoding” refers to the inherent property ofspecific sequences of nucleotides in a polynucleotide, such as a gene, acDNA, or an mRNA, to serve as templates for synthesis of other polymersand macromolecules in biological processes having either a definedsequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a definedsequence of amino acids and the biological properties resultingtherefrom.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked nucleic acid sequences arecontiguous and, where necessary to join two protein-coding regions, inthe same reading frame.

Peptide: A polymer in which the monomers are amino acid residues thatare joined together through amide bonds. The amino acids included in apeptide may be subject to post-translational modification (e.g.,glycosylation or phosphorylation). A “residue” refers to an amino acidor amino acid mimetic incorporated in a peptide by an amide bond oramide bond mimetic. A peptide has an amino terminal (N-terminal) end anda carboxy terminal (C-terminal) end. In some embodiments, a peptide canIn some embodiments, a peptide is at most 100 amino acids in length,such as at most 75 amino acids in length, such as at most 50 amino acidsin length or at most 40 amino acids in length for example.

Peptide Modifications: Synthetic embodiments of the peptides describedherein are also provided. For example, peptides can be modified by avariety of chemical techniques to produce derivatives having essentiallythe same activity as the unmodified peptides, and optionally havingother desirable properties. For example, carboxylic acid groups of thepeptide, whether carboxyl-terminal or side chain, can be provided in theform of a salt of a pharmaceutically-acceptable cation or esterified toform a C₁-C₁₆ ester, or converted to an amide of formula NR₁R₂ whereinR₁ and R2 are each independently H or C₁-C₁₆ alkyl, or combined to forma heterocyclic ring, such as a 5- or 6-membered ring Amino groups of thepeptide, whether amino-terminal or side chain, can be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or can be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the peptides of thisinvention to select and provide conformational constraints to thestructure that result in enhanced stability.

Each peptide of this disclosure is comprised of a sequence of aminoacids, which may be either L- and/or D-amino acids, naturally occurringand otherwise.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example, sodiumacetate or sorbitan monolaurate. In particular embodiments, suitable foradministration to a subject the carrier may be sterile, and/or suspendedor otherwise contained in a unit dosage form containing one or moremeasured doses of the composition suitable to elicit the desired immuneresponse. It may also be accompanied by medications for its use fortreatment purposes. The unit dosage form may be, for example, in asealed vial that contains sterile contents or a syringe for injectioninto a subject, or lyophilized for subsequent solubilization andadministration or in a solid or controlled release dosage.

Prime-boost immunization: An immunotherapy including administration ofmultiple immunogens over a period of time to elicit the desired immuneresponse.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity; the higher the percentage, themore similar the two sequences are. Homologs, orthologs, or variants ofa polypeptide will possess a relatively high degree of sequence identitywhen aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

Homologs and variants of a polypeptide are typically characterized bypossession of at least about 75%, for example, at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitycounted over the full length alignment with the amino acid sequence ofinterest. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.These sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

As used herein, reference to “at least 90% identity” (or similarlanguage) refers to “at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or even 100% identity” to a specified referencesequence.

Specifically bind: When referring to the formation of anantibody:antigen protein complex, or a protein:protein complex, refersto a binding reaction which determines the presence of a target protein,peptide, or polysaccharide (for example, a glycoprotein), in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated conditions, a particular antibody or proteinbinds preferentially to a particular target protein, peptide orpolysaccharide (such as an antigen present on the surface of a pathogen)and does not bind in a significant amount to other proteins orpolysaccharides present in the sample or subject. Specific binding canbe determined by standard methods. A first protein or antibodyspecifically binds to a target protein when the interaction has a K_(D)of less than 10⁻⁶ Molar, such as less than 10⁻⁷ Molar, less than 10⁻⁸Molar, less than 10⁻⁹, or even less than 10⁻¹⁰ Molar.

Subject: Living multicellular vertebrate organisms, a category thatincludes human and non-human mammals. In an example, a subject is ahuman. In an additional example, a subject is selected that is in needof inhibiting an ebolavirus infection. For example, the subject isuninfected and at risk of ebolavirus infection.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits a desired activity.

Vector: An entity containing a DNA or RNA molecule bearing a promoter(s)that is operationally linked to the coding sequence of an immunogenicprotein of interest and can express the coding sequence. Non-limitingexamples include a naked or packaged (lipid and/or protein) DNA, a nakedor packaged RNA, a subcomponent of a virus or bacterium or othermicroorganism that may be replication-incompetent, or a virus orbacterium or other microorganism that may be replication-competent. Avector is sometimes referred to as a construct. Recombinant DNA vectorsare vectors having recombinant DNA. A vector can include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector can also include one or more selectable markergenes and other genetic elements. Viral vectors are recombinant nucleicacid vectors having at least some nucleic acid sequences derived fromone or more viruses.

A non-limiting example of a DNA-based expression vector is pCDNA3.1,which can include includes a mammalian expression enhancer and promoter(such as a CMV promoter). Non-limiting examples of viral vectors includeadeno-associated virus (AAV) vectors as well as Poxvirus vector (e.g.,Vaccinia, MVA, avian Pox, or Adenovirus).

II. Filovirus GP Peptides

Isolated peptides containing fragments of filovirus GP proteins aredisclosed herein that can be used to induce an immune response in asubject that neutralizs filovirus infection (such as Zaire ebolavirusinfection. As discussed in the Examples, the isolated peptides containantigenic sites of the filovirus GP that are targeted by neutralizingantibodies and are shown to induce an immune response in a subject thatneutralizing filovirus infection (such as Zaire ebolavirus infection).

In several embodiments, the isolated peptides contain fragments of anebolavirus GP, such as GP from one of Zaire ebolavirus, Bundibugyoebolavirus, Sudan ebolavirus, or Tai Forest ebolavirus. Exemplaryebolavirus GP protein sequences are set forth herein as SEQ ID NOs:34-41. In some embodiments, the isolated peptides contain fragments of aMarburg margburgvirus GP An exemplary Marburg margburgvirus GP proteinsequence is set forth herein as SEQ ID NO: 42. In further embodiments,the isolated peptide contains a fragment of a GP from another Filovirusspecies.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of 10-100 consecutive amino acids or longer(such as 10-15, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40, 20-50, 30-50,40-50, 10-75, 20-75, 20-75, 20-75, 30-75, 40-75, 50-75, or 75-100consecutive amino acids) from a native filovirus GP sequence, such as aGP sequence set forth as any one of SEQ ID NOs: 34-42. In someembodiments the isolated peptide is no more than 100 amino acids inlength, such as no more than 75, or no more than 50, or no more than 40amino acids in length.

The isolated peptide includes the amino acid sequence of an antigenicsite of the filovirus GP. For example, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence of anyone of the antigenic sites of Zaire ebolavirus as set forth in FIG. 20,such as Antigenic site II.1, 11.2, 11.3, III.1, 111.2, IV.1, IV.2, IV.3,IV.4, IV.5, V.1, V.2, V.3, V.4, V.5, V.6, V.7, V.8, V.9. V.10, V.11, orVI. The sequences of antigenic sites shown in FIG. 20 correspond to theZaire ebolavirus Mayinga GP sequence set forth as SEQ ID NO: 35. Due tothe sequence homology of GP across filoviruses (see the filovirus GPsequence alignment provided in FIGS. 19A-19C), the antigenic sitesprovided in FIG. 20 can readily be identified in other filovirus GPsequences, for example, a GP from any of Zaire ebolavirus, Bundibugyoebolavirus, Sudan ebolavirus, or Tai Forest ebolavirus, or Marburgmargburgvirus, such as a GP protein sequence is set forth any one of SEQID NOs: 34-42, or other Filovirus strains

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of the antigenicsite VI residues from a filovirus GP, such as an ebolavirus GP orMarburgvirus GP. In some embodiments, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence setforth as SEQ ID NO: 9: KNITDKIX₁QIIHDFX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWW, whereinX₁ is D or N, X₂ is V or I, X₃ is K or N, X₄ is T, P, or N, X₅ is D orN, X₆ is G, T, D, or N, X₇ is D or N, X₈ is N, D, or G, and X₉ is D orS, wherein the peptide is no more than 100 (such as no more than 75, 50,40, or 30) amino acids in length and induces a neutralizing immuneresponse to filovirus (such as ebolavirus) in a subject. SEQ ID NO: 9 isa consensus amino acid sequence encompassing the amino acids ofantigenic site VI from GP of Zaire ebolavirus (Mayinga, Kikwit, andMakona), Bundibugyo ebolavirus, Sudan ebolavirus, and Tai Forestebolavirus. For example, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence set forth as anyone of SEQ ID NOs: 10-13. In further embodiments, the isolated peptidecomprises, consists essentially of, or consists of the amino acidsequence of the antigenic site VI from a Margurg Marburgvirus, such asthe Antigenic site VI sequence set forth as SEQ ID NO: 53.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence set forth as SEQID NO: 7: KNITDKIX₁QIIHDFX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWWT, wherein: X₁ is D orN, X₂ is V and I, X₃ is K or N, X₄ is T, P, or N, X₅ is D or N, X₆ is G,T, D, or N, X₇ is D or N, X₈ is N, D, or G; and X₉ is D or S, whereinthe peptide is no more than 100 (such as no more than 75, 50, 40, or 30)amino acids in length and induces a neutralizing immune response tofilovirus (such as ebolavirus) in a subject. SEQ ID NO: 7 is a consensusamino acid sequence encompassing the amino acids of antigenic site VIfrom GP of Zaire ebolavirus (Mayinga, Kikwit, and Makona), Bundibugyoebolavirus, Sudan ebolavirus, and Tai Forest ebolavirus. For example,the isolated peptide comprises, consists essentially of, or consists ofthe amino acid sequence set forth as any one of SEQ ID NOs: 4, or 14-16.In further embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of the antigenicsite VI from a Margurg Marburgvirus, such as the Antigenic site VIsequence set forth as SEQ ID NO: 54.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of the antigenicsite VI.1 residues from a filovirus GP, such as an ebolavirus GP orMarburgvirus GP. In some embodiments, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence setforth as SEQ ID NO: 6 (FX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWWT, wherein: X₂ isselected from V and I; X₃ from K and N; X₄ from T, P, and N; X₅ and X₇from D and N; X₆ from G, T, D, and N; X₈ from N, D, and G; and X₉ from Dand S, wherein the peptide is no more than 100 (such as no more than 75,50, 40, or 30) amino acids in length and induces a neutralizing immuneresponse to filovirus (such as ebolavirus) in a subject. SEQ ID NO: 6 isa consensus amino acid sequence encompassing a C-terminal portion ofantigenic site VI.1 from GP of Zaire ebolavirus (Mayinga, Kikwit, andMakona), Bundibugyo ebolavirus, Sudan ebolavirus, and Tai Forestebolavirus. For example, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence set forth as anyone of SEQ ID NOs: 5, or residues 14-30 of SEQ ID NOs: 14-16 or 54.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of the antigenicsite IV.1 residues from a filovirus GP, such as an ebolavirus GP orMarburgvirus GP. In some embodiments, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence setforth as any one of SEQ ID NOs: 17-20, wherein the peptide is no morethan 100 (such as no more than 75, 50, 40, or 30) amino acids in lengthand induces a neutralizing immune response to filovirus (such asebolavirus) in a subject. SEQ ID NOs: 17-20 encompass the amino acids ofantigenic site IV.1 from GP of Zaire ebolavirus (Mayinga, Kikwit, andMakona), Bundibugyo ebolavirus, Sudan ebolavirus, and Tai Forestebolavirus or other Filoviruses. In further embodiments, the isolatedpeptide comprises, consists essentially of, or consists of the aminoacid sequence of the antigenic site VI from a Margurg Marburgvirus, suchas the Antigenic site IV.1 sequence set forth as SEQ ID NO: 58.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of the antigenicsite V.1 residues from a filovirus GP, such as an ebolavirus GP orMarburgvirus GP. In some embodiments, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence setforth as any one of SEQ ID NOs: 21-25, wherein the peptide is no morethan 100 (such as no more than 75, 50, 40, or 30) amino acids in lengthand induces a neutralizing immune response to filovirus (such asebolavirus) in a subject. SEQ ID NOs: 21-25 encompass the amino acids ofantigenic site V.1 from GP of Zaire ebolavirus (Mayinga, Kikwit, andMakona), Bundibugyo ebolavirus, Sudan ebolavirus, and Tai Forestebolavirus or other Filoviruses. In further embodiments, the isolatedpeptide comprises, consists essentially of, or consists of the aminoacid sequence of the antigenic site VI from a Margurg Marburgvirus, suchas the Antigenic site V.1 sequence set forth as SEQ ID NO: 59.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of the antigenicsite V.6 residues from a filovirus GP, such as an ebolavirus GP orMarburgvirus GP. In some embodiments, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence setforth as any one of SEQ ID NOs: 2, 26-28, or 43-46 wherein the peptideis no more than 100 (such as no more than 75, 50, 40, or 30) amino acidsin length and induces a neutralizing immune response to filovirus (suchas ebolavirus) in a subject. SEQ ID NOs: 2, 26-28, and 43-46 encompassthe amino acids of antigenic site V.6 from GP of Zaire ebolavirus(Mayinga, Kikwit, and Makona), Bundibugyo ebolavirus, Sudan ebolavirus,and Tai Forest ebolavirus or other Filoviruses. In further embodiments,the isolated peptide comprises, consists essentially of, or consists ofthe amino acid sequence of the antigenic site VI from a MargurgMarburgvirus, such as the Antigenic site V.6 sequence set forth as SEQID NO: 56.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of the antigenicsite V.7 residues from a filovirus GP, such as an ebolavirus GP orMarburgvirus GP. In some embodiments, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence setforth as any one of SEQ ID NOs: 3 or 32-34, wherein the peptide is nomore than 100 (such as no more than 75, 50, 40, or 30) amino acids inlength and induces a neutralizing immune response to filovirus (such asebolavirus) in a subject. SEQ ID NOs: 3 and 32-34 encompass the aminoacids of antigenic site V.7 from GP of Zaire ebolavirus (Mayinga,Kikwit, and Makona), Bundibugyo ebolavirus, Sudan ebolavirus, and TaiForest ebolavirus or other Filoviruses. In further embodiments, theisolated peptide comprises, consists essentially of, or consists of theamino acid sequence of the antigenic site V.7 from a MargurgMarburgvirus, such as the Antigenic site VI sequence set forth as SEQ IDNO: 55.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of the antigenicsite V.10 residues from a filovirus GP, such as an ebolavirus GP orMarburgvirus GP. In some embodiments, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence setforth as any one of SEQ ID NOs: 29-31, wherein the peptide is no morethan 100 (such as no more than 75, 50, 40, or 30) amino acids in lengthand induces a neutralizing immune response to filovirus (such asebolavirus) in a subject. SEQ ID NOs: 29-31 encompass the amino acids ofantigenic site V.10 from GP of Zaire ebolavirus (Mayinga, Kikwit, andMakona), Bundibugyo ebolavirus, Sudan ebolavirus, and Tai Forestebolavirus or other Filoviruses. In further embodiments, the isolatedpeptide comprises, consists essentially of, or consists of the aminoacid sequence of the antigenic site V.10 from a Margurg Marburgvirus,such as the Antigenic site VI sequence set forth as SEQ ID NO: 60.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of a portion ofantigenic site V residues from a filovirus GP, such as an ebolavirus GPor Marburgvirus GP. In some embodiments, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence setforth as SEQ ID NO: 8: TX₁₀EDHKIMASENSSAMVQVHSQGRX₁₁AAVSH wherein: X₁₀is T or N; and X₁₁ is E or K, wherein the peptide is no more than 100(such as no more than 75, 50, 40, or 30) amino acids in length andinduces a neutralizing immune response to filovirus (such as ebolavirus)in a subject. SEQ ID NO: 8 is a consensus amino acid sequence includinga portion of antigenic site V from GP of Zaire ebolavirus (Mayinga,Kikwit, and Makona), Bundibugyo ebolavirus, Sudan ebolavirus, and TaiForest ebolavirus. For example, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence set forth as SEQID NO: 1.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of antigenic siteII.1 residues from a filovirus GP, such as an ebolavirus GP orMarburgvirus GP. In some embodiments, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence setforth as any one of SEQ ID NOs: 47-50, wherein the peptide is no morethan 100 (such as no more than 75, 50, 40, or 30) amino acids in lengthand induces a neutralizing immune response to filovirus (such asebolavirus) in a subject. SEQ ID NOs: 47-50 encompass the amino acids ofantigenic site II.1 from GP of Zaire ebolavirus (Mayinga, Kikwit, andMakona), Bundibugyo ebolavirus, Sudan ebolavirus, and Tai Forestebolavirus or other Filoviruses. In further embodiments, the isolatedpeptide comprises, consists essentially of, or consists of the aminoacid sequence of the antigenic site VI from a Margurg Marburgvirus, suchas the Antigenic site II.1 sequence set forth as SEQ ID NO: 61.

In some embodiments, the isolated peptide comprises, consistsessentially of, or consists of the amino acid sequence of the antigenicsite IV.2 residues from a filovirus GP, such as an ebolavirus GP orMarburgvirus GP. In some embodiments, the isolated peptide comprises,consists essentially of, or consists of the amino acid sequence setforth as any one of SEQ ID NOs: 51-52, wherein the peptide is no morethan 100 (such as no more than 75, 50, 40, or 30) amino acids in lengthand induces a neutralizing immune response to filovirus (such asebolavirus) in a subject. SEQ ID NOs: 51-51 encompass the amino acids ofantigenic site IV.2 from GP of Zaire ebolavirus (Mayinga, Kikwit, andMakona), Bundibugyo ebolavirus, Sudan ebolavirus, and Tai Forestebolavirus or other Filoviruses. In further embodiments, the isolatedpeptide comprises, consists essentially of, or consists of the aminoacid sequence of the antigenic site VI from a Margurg Marburgvirus, suchas the Antigenic site IV.2 sequence set forth as SEQ ID NO: 62.

Any of the isolated peptides disclosed herein can be conjugated to acarrier molecule, for example, to enhance the immune response in asubject to the peptide. The peptide can be directly conjugated to thecarrier or indirectly via a linker.

In some examples, the peptide and the carrier are linked by a linkerbetween a lysine amino acid residue present on the carrier protein and acysteine amino acid residue fused (by a peptide bond) to the C-terminalresidue of the peptide.

Suitable linkers include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers or peptidelinkers. For an immunogenic conjugate from two or more constituents,each of the constituents will contain the necessary reactive groups.Representative combinations of such groups are amino with carboxyl toform amide linkages or carboxy with hydroxyl to form ester linkages oramino with alkyl halides to form alkylamino linkages or thiols withthiols to form disulfides or thiols with maleimides or alkylhalides toform thioethers. Hydroxyl, carboxyl, amino and other functionalities,where not present may be introduced by known methods. Likewise, a widevariety of linking groups may be employed. In some cases, the linkinggroup can be designed to be either hydrophilic or hydrophobic in orderto enhance the desired binding characteristics of the peptide and thecarrier. The covalent linkages should be stable relative to the solutionconditions under which the conjugate is subjected.

In some embodiments, the linkers may be joined to the constituent aminoacids through their side chains (such as through a disulfide linkage tocysteine) or to the alpha carbon, amino, and/or carboxyl groups of theterminal amino acids. In some embodiments, the linker, the peptide, andthe carrier can be encoded as a single peptide such that the peptide andthe carrier are joined by peptide bonds.

The procedure for attaching a carrier molecule to a peptide variesaccording to the chemical structure of the molecules. Peptides typicallycontain a variety of functional groups; for example, carboxylic acid(COOH), free amine (—NH₂) or sulfhydryl (—SH) groups, which areavailable for reaction with a suitable functional group on a peptide.Alternatively, the peptide is derivatized to expose or attach additionalreactive functional groups. The derivatization may involve attachment ofany of a number of linker molecules such as those available from PierceChemical Company, Rockford, Ill.

In some embodiments, a sulfosuccinimidyl (4-iodoacetyl)aminobenzoate(Sulfo-SIAB) linker is used to link the peptide to carrier. In someembodiments an m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)linker is used to attach the peptide to carrier.

Any specific combination of peptide and carrier may be selected from thespecific peptides and carrier that are listed herein.

It can be advantageous to produce conjugates in which more than onepeptide as described herein is conjugated to a single carrier protein.In several embodiments, the conjugation of multiple peptides to a singlecarrier protein is possible because the carrier protein has multiplelysine or cysteine side-chains that can serve as sites of attachment.The amount of peptide reacted with the amount of carrier may varydepending upon the specific peptide and the carrier. In someembodiments, from 1 to 30, such as about 1, about 5, about 10, about 15,about 20, or about 30 peptides, or more, can be linked to each carrierprotein molecule. In some embodiments (such as when KLH is used as acarrier, from 1 to 1000, such as about 10, about 20, about 30, about 40,about 50, about 100, about 200, about 300, about 400, about 500, about700, or about 1000 peptides can be linked to each carrier proteinmolecule. “About” in this context refers to plus or minus 5% whenmeasuring an average number of X peptide molecules per carrier moleculein the conjugate.

Examples of suitable carriers are those that can increase theimmunogenicity of the conjugate and/or elicit antibodies against thecarrier which are diagnostically, analytically, and/or therapeuticallybeneficial. Useful carriers include polymeric carriers, which can benatural, recombinantly produced, semi-synthetic or synthetic materialscontaining one or more amino groups, such as those present in a lysineamino acid residue present in the carrier, to which a reactant moietycan be attached. Carriers that fulfill these criteria are available(see, for example, Fattom et al., Infect. Immun. 58:2309-12, 1990; Deviet al., PNAS 88:7175-79, 1991; Szu et al., Infect. Immun. 59:4555-61,1991; Szu et al., J. Exp. Med. 166:1510-24, 1987; and Pavliakova et al.,Infect. Immun. 68:2161-66, 2000). A carrier can be useful even if theantibody that it elicits is not of benefit by itself.

Specific, non-limiting examples of suitable polypeptide carriersinclude, but are not limited to, natural, semi-synthetic or syntheticpolypeptides or proteins from bacteria or viruses. In one embodiment,bacterial products for use as carriers include bacterial toxins.Bacterial toxins include bacterial products that mediate toxic effects,inflammatory responses, stress, shock, chronic sequelae, or mortality ina susceptible host. Specific, non-limiting examples of bacterial toxinsinclude, but are not limited to: B. anthracis PA (for example, asencoded by bases 143779 to 146073 of GENBANK® Accession No. NC 007322);B. anthracis LF (for example, as encoded by the complement of bases149357 to 151786 of GENBANK® Accession No. NC 007322); bacterial toxinsand toxoids, such as tetanus toxin/toxoid (for example, as described inU.S. Pat. Nos. 5,601,826 and 6,696,065); diphtheria toxin/toxoid (forexample, as described in U.S. Pat. Nos. 4,709,017 and 6,696,065), suchas tetanus toxin heavy chain C fragment; P. aeruginosa exotoxin/toxoid(for example, as described in U.S. Pat. Nos. 4,428,931, 4,488,991 and5,602,095); pertussis toxin/toxoid (for example, as described in U.S.Pat. Nos. 4,997,915, 6,399,076 and 6,696,065); and C. perfringensexotoxin/toxoid (for example, as described in U.S. Pat. Nos. 5,817,317and 6,403,094) C. difficile toxin B or A, or analogs or mimetics of andcombinations of two or more thereof. Viral proteins, such as hepatitis Bsurface antigen (for example, as described in U.S. Pat. Nos. 5,151,023and 6,013,264) and core antigen (for example, as described in U.S. Pat.Nos. 4,547,367 and 4,547,368) can also be used as carriers, as well asproteins from higher organisms such as keyhole limpet hemocyanin (KLH),horseshoe crab hemocyanin, Concholepas Concholepas Hemocyanin (CCH),Ovalbumin (OVA), edestin, mammalian serum albumins (such as bovine serumalbumin), and mammalian immunoglobulins. In some examples, the carrieris bovine serum albumin.

In some embodiments, the carrier is selected from one of: Keyhole LimpetHemocyanin (KLH), tetanus toxoid, tetanus toxin heavy chain C fragment,diphtheria toxoid, diphtheria toxin variant CRM197, or H influenzaprotein D (HiD). CRM197 is a genetically detoxified form of diphtheriatoxin; a single mutation at position 52, substituting glutamic acid forglycine, causes the ADP-ribosyltransferase activity of the nativediphtheria toxin to be lost. For description of protein carriers forvaccines, see Pichichero, Protein carriers of conjugate vaccines:characteristics, development, and clinical trials, Hum VaccinImmunother., 9: 2505-2523, 2013, which is incorporated by referenceherein in its entirety).

Following conjugation of the peptide to the carrier protein, theconjugate can be purified by appropriate techniques. One goal of thepurification step is to separate the unconjugated peptide or carrierfrom the conjugate. One method for purification, involvingultrafiltration in the presence of ammonium sulfate, is described inU.S. Pat. No. 6,146,902. Alternatively, the conjugates can be purifiedaway from unconjugated peptide or carrier by any number of standardtechniques including, for example, size exclusion chromatography,density gradient centrifugation, hydrophobic interaction chromatography,or ammonium sulfate fractionation. See, for example, Anderson et al., J.Immunol. 137:1181-86, 1986 and Jennings & Lugowski, J. Immunol.127:1011-18, 1981. The compositions and purity of the conjugates can bedetermined by GLC-MS and MALDI-TOF spectrometry, for example.

In several embodiments, the disclosed immunogenic conjugates can beformulated into an immunogenic composition (such as vaccines), forexample by the addition of a pharmaceutically acceptable carrier and/oradjuvant.

III. Polynucleotides and Expression

Polynucleotides encoding the disclosed peptides are also provided. Thesepolynucleotides include DNA, cDNA and RNA sequences which encode thepeptide. The genetic code can be used to construct a variety offunctionally equivalent nucleic acids, such as nucleic acids that differin sequence but which encode the same protein sequence.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises areknown (see, e.g., Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,through supplement 104, 2013).

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

The polynucleotides encoding a disclosed peptide can include arecombinant DNA which is incorporated into a vector into an autonomouslyreplicating plasmid or virus or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (such as a cDNA)independent of other sequences. The nucleotides can be ribonucleotides,deoxyribonucleotides, or modified forms of either nucleotide. The termincludes single and double forms of DNA.

Polynucleotide sequences encoding a disclosed peptide can be operativelylinked to expression control sequences. An expression control sequenceoperatively linked to a coding sequence is ligated such that expressionof the coding sequence is achieved under conditions compatible with theexpression control sequences. The expression control sequences include,but are not limited to, appropriate promoters, enhancers, transcriptionterminators, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signals for introns, maintenance of the correct readingframe of that gene to permit proper translation of mRNA, and stopcodons.

DNA sequences encoding the disclosed peptide can be expressed in vitroby DNA transfer into a suitable host cell. The cell may be prokaryoticor eukaryotic. The term also includes any progeny of the subject hostcell. It is understood that all progeny may not be identical to theparental cell since there may be mutations that occur duringreplication. Methods of stable transfer, meaning that the foreign DNA iscontinuously maintained in the host, are known in the art.

Hosts can include microbial, yeast, insect and mammalian organisms.Methods of expressing DNA sequences having eukaryotic or viral sequencesin prokaryotes are well known in the art. Non-limiting examples ofsuitable host cells include bacteria, archea, insect, fungi (forexample, yeast), plant, and animal cells (for example, mammalian cells,such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C₁₂₉ cells, 293 cells, Neurospora, and immortalized mammalianmyeloid and lymphoid cell lines. Techniques for the propagation ofmammalian cells in culture are well-known (see, e.g., Helgason andMiller (Eds.), 2012, Basic Cell Culture Protocols (Methods in MolecularBiology), 4^(th) Ed., Humana Press). Examples of commonly used mammalianhost cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, andCOS cell lines, although cell lines may be used, such as cells designedto provide higher expression, desirable glycosylation patterns, or otherfeatures. In some embodiments, the host cells include HEK293 cells orderivatives thereof, such as GnTI^(−/−) cells (ATCC® No. CRL-3022), orHEK-293F cells.

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques. In some embodiments, if the host isprokaryotic, such as, but not limited to, E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or viral vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding a disclosedantigen, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, ViralExpression Vectors, Springer press, Muzyczka ed., 2011). Appropriateexpression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines can be utilized.

Modifications can be made to a nucleic acid encoding a disclosed peptidewithout diminishing its biological activity. Some modifications can bemade to facilitate the cloning, expression, or incorporation of thepeptide into a fusion protein. Non-limiting examples of suchmodifications include termination codons, a methionine added at theamino terminus to provide an initiation site, additional amino acidsplaced on either terminus to create conveniently located restrictionsites, or additional amino acids (such as poly His) to aid inpurification steps.

IV. Viral Vectors

A nucleic acid molecule encoding a disclosed peptide can be included ina viral vector, for example, for expression of the immunogen in a hostcell, or for immunization of a subject as disclosed herein. In someembodiments, the viral vectors are administered to a subject as part ofa prime-boost immunization. In several embodiments, the viral vectorsused in a prime-boost immunization protocol to prime an immune responseto ebolavirus GP or boost an immune response to ebolavirus GP.

In several examples, the viral vector can be replication-competent. Forexample, the viral vector can have a mutation in the viral genome thatdoes not inhibit viral replication in host cells. The viral vector alsocan be conditionally replication-competent. In other examples, the viralvector is replication-deficient in host cells.

A number of viral vectors have been constructed that can be used toexpress the disclosed antigens, including polyoma, i.e., SV40 (Madzak etal., 1992, J. Gen. Virol., 73:15331536), adenovirus (Berkner, 1992, Cur.Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, BioTechniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412;Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584;Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl.Acids Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. GeneTher., 1:241-256), vaccinia virus (Mackett et al., 1992, Biotechnology,24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top.Microbiol. Immunol., 158:91-123; On et al., 1990, Gene, 89:279-282),herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top.Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol.,66:29522965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield etal., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem.Pharmacol., 40:2189-2199), Sindbis viruses (H. Herweijer et al., 1995,Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol.11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA93:11371-11377) and retroviruses of avian (Brandyopadhyay et al., 1984,Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol.,66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol.,158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al.,1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol.,54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors are also known in the art, and may be obtained fromcommercial sources (such as PharMingen, San Diego, Calif.; ProteinSciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

VI. Immunogenic Compositions

Immunogenic compositions comprising a disclosed peptide (for example,linked to a carrier) or a nucleic acid molecule or vector encoding thepeptide and a pharmaceutically acceptable carrier are also provided.Such compositions can be administered to subjects by a variety ofadministration modes, for example, intramuscular, subcutaneous,intravenous, intra-arterial, intra-articular, intraperitoneal, orparenteral routes. Actual methods for preparing administrablecompositions are described in more detail in such publications asRemingtons Pharmaceutical Sciences, 19^(th) Ed., Mack PublishingCompany, Easton, Pa., 1995.

The peptide (for example, linked to a carrier) or a nucleic acidmolecule or vector encoding the peptide can be formulated withpharmaceutically acceptable carriers to help retain biological activitywhile also promoting increased stability during storage within anacceptable temperature range. Potential carriers include, but are notlimited to, physiologically balanced culture medium, phosphate buffersaline solution, water, emulsions (e.g., oil/water or water/oilemulsions), various types of wetting agents, cryoprotective additives orstabilizers such as proteins, peptides or hydrolysates (e.g., albumin,gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g.,sodium glutamate), or other protective agents. The resulting aqueoussolutions may be packaged for use as is or lyophilized. Lyophilizedpreparations are combined with a sterile solution prior toadministration for either single or multiple dosing.

Formulated compositions, especially liquid formulations, may contain abacteriostat to prevent or minimize degradation during storage,including but not limited to effective concentrations (usually 1% w/v)of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben,and/or propylparaben. A bacteriostat may be contraindicated for somepatients; therefore, a lyophilized formulation may be reconstituted in asolution either containing or not containing such a component.

The immunogenic compositions of the disclosure can contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate.

The immunogenic composition may optionally include an adjuvant toenhance an immune response of the host. Suitable adjuvants are, forexample, toll-like receptor agonists, alum, AlPO4, alhydrogel, Lipid-Aand derivatives or variants thereof, oil-emulsions, saponins, neutralliposomes, liposomes containing the vaccine and cytokines, non-ionicblock copolymers, and chemokines. Non-ionic block polymers containingpolyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POEblock copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.) mayalso be used as an adjuvant (Newman et al., 1998, Critical Reviews inTherapeutic Drug Carrier Systems 15:89-142). These adjuvants have theadvantage in that they help to stimulate the immune system in anon-specific way, thus enhancing the immune response to a pharmaceuticalproduct.

In some embodiments, the immunogenic composition can be provided as asterile composition. The immunogenic composition typically contains aneffective amount of a disclosed peptide (for example, linked to acarrier) or a nucleic acid molecule or vector encoding the peptide, andcan be prepared by conventional techniques. Typically, the amount of adisclosed peptide (for example, linked to a carrier) or a nucleic acidmolecule or vector encoding the peptide in each dose of the immunogeniccomposition is selected as an amount which elicits an immune responsewithout significant, adverse side effects. In some embodiments, theimmunogenic composition can be provided in unit dosage form for use toelicit an immune response in a subject, for example, to preventebolavirus infection in the subject. A unit dosage form contains asuitable single preselected dosage for administration to a subject, orsuitable marked or measured multiples of two or more preselected unitdosages, and/or a metering mechanism for administering the unit dose ormultiples thereof. In other embodiments, the composition furtherincludes an adjuvant.

VII. Methods of Inducing an Immune Response

An immunogenic composition comprising a disclosedfi/ovirus GP (e.g.,Zaire ebolavirus GP) peptide, a nucleic acid molecule (such as an RNAmolecule) encoding a disclosedfi/ovirus GP (e.g., Zaire ebolavirus GP)peptide, vector including the nucleic acid molecule, or immunogeniccomposition, can be administered to a subject to induce an immuneresponse to filovirus GP (e.g., Zaire ebolavirus GP) in the subject. Ina particular example, the subject is a human. The immune response can bea protective immune response, for example a response that inhibitssubsequent infection with a filovirus (such as a Zaire ebolavirus).Elicitation of the immune response can also be used to treat or inhibitinfection and illnesses associated with a filovirus (such as a Zaireebolavirus).

A subject can be selected for immunization that has, or is at risk fordeveloping infection or illness associated with a filovirus (such as aZaire ebolavirus), for example because of exposure or the possibility ofexposure to a filovirus (such as a Zaire ebolavirus).

Typical subjects intended for administration of the immunogeniccomposition include humans, as well as non-human primates and otheranimals. To identify relevant subjects, accepted screening methods areemployed to determine risk factors associated with a targeted orsuspected disease or condition, or to determine the status of anexisting disease or condition in a subject. These screening methodsinclude, for example, conventional work-ups to determine environmental,familial, occupational, and other such risk factors that may beassociated with the targeted or suspected disease or condition, as wellas diagnostic methods, such as various ELISA and other immunoassaymethods to detect and/or characterize a filovirus (such as a Zaireebolavirus) infection. These and other routine methods allow theclinician to select patients in need of therapy. In accordance withthese methods and principles, the immunogenic composition can beadministered according to the teachings herein, or other conventionalmethods, as an independent prophylaxis or treatment program, or as afollow-up, adjunct or coordinate treatment regimen to other treatments.

The administration of the immunogenic composition can be forprophylactic or therapeutic purpose. When provided prophylactically, theimmunogenic composition can be provided in advance of any symptom, forexample, in advance of infection. The prophylactic administration servesto prevent or ameliorate any subsequent infection. In some embodiments,the methods can involve selecting a subject at risk for contractingfilovirus infection (e.g., Zaire ebolavirus infection), andadministering an effective amount of the immunogenic composition to thesubject. The immunogenic composition can be provided prior to theanticipated exposure to filovirus infection (e.g., Zaire ebolavirusinfection) so as to attenuate the anticipated severity, duration orextent of an infection and/or associated disease symptoms, afterexposure or suspected exposure to the virus, or after the actualinitiation of an infection.

The immunogenic composition is provided to the subject in an amounteffective to induce or to enhance an immune response against filovirusGP (e.g., Zaire ebolavirus GP) in the subject, preferably a human. Theactual dosage of the immunogenic composition will vary according tofactors such as the disease indication and particular status of thesubject (for example, the subject's age, size, fitness, extent ofsymptoms, susceptibility factors, and the like), time and route ofadministration, other drugs or treatments being administeredconcurrently, as well as the specific pharmacology of the compositionfor eliciting the desired activity or biological response in thesubject. Dosage regimens can be adjusted to provide an optimumprophylactic or therapeutic response.

An immunogenic composition including one or more of the disclosedimmunogens can be used in coordinate (or prime-boost) vaccinationprotocols or combinatorial formulations. In certain embodiments, novelcombinatorial immunogenic compositions and coordinate immunizationprotocols employ separate immunogens or formulations, each directedtoward eliciting an anti-viral immune response, such as an immuneresponse to filovirus GP (e.g., Zaire ebolavirus GP). Separateimmunogenic compositions that elicit the anti-viral immune response canbe combined in a polyvalent immunogenic composition administered to asubject in a single immunization step, or they can be administeredseparately (in monovalent immunogenic compositions) in a coordinate (orprime-boost) immunization protocol.

There can be several boosts, and each boost can be a different disclosedimmunogen. In some examples that the boost may be the same immunogen asanother boost, or the prime. The prime and boost can be administered asa single dose or multiple doses, for example two doses, three doses,four doses, five doses, six doses or more can be administered to asubject over days, weeks or months. Multiple boosts can also be given,such one to five (e.g., 1, 2, 3, 4 or 5 boosts), or more. Differentdosages can be used in a series of sequential immunizations. For examplea relatively large dose in a primary immunization and then a boost withrelatively smaller doses.

In some embodiments, the boost can be administered about two, aboutthree to eight, or about four, weeks following the prime, or aboutseveral months after the prime. In some embodiments, the boost can beadministered about 5, about 6, about 7, about 8, about 10, about 12,about 18, about 24, months after the prime, or more or less time afterthe prime. Periodic additional boosts can also be used at appropriatetime points to enhance the subject's “immune memory.” The adequacy ofthe vaccination parameters chosen, e.g., formulation, dose, regimen andthe like, can be determined by taking aliquots of serum from the subjectand assaying antibody titers during the course of the immunizationprogram. In addition, the clinical condition of the subject can bemonitored for the desired effect, e.g., inhibition of filovirusinfection (e.g., Zaire ebolavirus infection) or improvement in diseasestate (e.g., reduction in viral load). If such monitoring indicates thatvaccination is sub-optimal, the subject can be boosted with anadditional dose of immunogenic composition, and the vaccinationparameters can be modified in a manner expected to potentiate the immuneresponse.

In some embodiments, the prime-boost method can include DNA-prime andprotein-boost vaccination protocol to a subject. The method can includetwo or more administrations of the nucleic acid molecule or the protein.

For peptide therapeutics, typically, each human dose will comprise1-1000 ng of protein, such as from about 1 ng to about 100 μg, forexample, from about 1 ng to about 50 μg, such as about 1 μg, about 2 μg,about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30μg, about 40 μg, or about 50 μg.

The amount utilized in an immunogenic composition is selected based onthe subject population (e.g., infant or elderly). An optimal amount fora particular composition can be ascertained by standard studiesinvolving observation of antibody titers and other responses insubjects. It is understood that an effective amount of a disclosedimmunogenic composition can include an amount that is ineffective ateliciting an immune response by administration of a single dose, butthat is effective upon administration of multiple dosages, for examplein a prime-boost administration protocol.

Upon administration of the immunogenic composition, the immune system ofthe subject typically responds to the immunogenic composition byproducing antibodies specific for viral protein. Such a responsesignifies that an immunologically effective dose was delivered to thesubject.

In some embodiments, the antibody response of a subject will bedetermined in the context of evaluating effective dosages/immunizationprotocols. In most instances it will be sufficient to assess theantibody titer in serum or plasma obtained from the subject. Decisionsas to whether to administer booster inoculations and/or to change theamount of the therapeutic agent administered to the individual can be atleast partially based on the antibody titer level. The antibody titerlevel can be based on, for example, an immunobinding assay whichmeasures the concentration of antibodies in the serum which bind to anantigen including, for example, filovirus GP (e.g., Zaire ebolavirusGP).

Filovirus infection (e.g., Zaire ebolavirus infection) does not need tobe completely eliminated or reduced or prevented for the methods to beeffective. For example, elicitation of the immune response can reduce orinhibit infection with the Filovirus (e.g., Zaire ebolavirus) by adesired amount, for example, by at least 10%, at least 20%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or even at least 100% (elimination or prevention ofdetectable infected cells), as compared to infection with the Filovirus(e.g., Zaire ebolavirus) in the absence of the immunization.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid.Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. Nos.5,593,972 and 5,817,637 (which describe operably linking a nucleic acidsequence encoding an antigen to regulatory sequences enablingexpression). U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Protective immunity has beengenerated in a variety of experimental models of infection, includingtoxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ asthe delivery vehicle for antigens (Mowat and Donachie, Immunol. Today12:383, 1991). Doses of antigen as low as 1 jag encapsulated in ISCOMS™have been found to produce Class I mediated CTL responses (Takahashi etal., Nature 344:873, 1990).

In some embodiments, a plasmid DNA vaccine is used to express adisclosed filovirus GP peptide (e.g., Zaire ebolavirus GP peptide) in asubject. For example, a nucleic acid molecule encoding a disclosedfilovirus GP peptide (e.g., Zaire ebolavirus GP peptide) can beadministered to a subject to induce an immune response to filovirus GP(e.g., Zaire ebolavirus GP).

In another approach, a disclosed filovirus GP peptide (e.g., Zaireebolavirus GP peptide) can be expressed by attenuated viral hosts orvectors or bacterial vectors. Recombinant vaccinia virus,adeno-associated virus (AAV), herpes virus, retrovirus, cytogmeglo virusor other viral vectors can be used to express the peptide or protein,thereby eliciting a CTL response. For example, vaccinia vectors andmethods useful in immunization protocols are described in U.S. Pat. No.4,722,848. BCG (Bacillus Calmette Guerin) provides another vector forexpression of the peptides (see Stover, Nature 351:456-460, 1991). Thesepeptides can also be used in combination or with vaccines against otherpathogens.

In one embodiment, a nucleic acid encoding a disclosed filovirus GPpeptide (e.g., Zaire ebolavirus GP peptide) is introduced directly intocells to induce the immune response. For example, the nucleic acid canbe loaded onto gold microspheres by standard methods and introduced intothe skin by a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleicacids can be “naked,” consisting of plasmids under control of a strongpromoter. Typically, the DNA is injected into muscle, although it canalso be injected directly into other sites. Dosages for injection areusually around 0.5 μg/kg to about 50 mg/kg, and typically are about0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Pat. No. 5,589,466).

In another embodiment, an mRNA-based immunization protocol can be usedto deliver a nucleic acid encoding a disclosed filovirus GP peptide(e.g., Zaire ebolavirus GP peptide) directly into cells. In someembodiments, nucleic acid-based vaccines based on mRNA may provide apotent alternative to the previously mentioned approaches. mRNA vaccinespreclude safety concerns about DNA integration into the host genome andcan be directly translated in the host cell cytoplasm. Moreover, thesimple cell-free, in vitro synthesis of RNA avoids the manufacturingcomplications associated with viral vectors. Two exemplary forms ofRNA-based vaccination that can be used to deliver a nucleic acidencoding a disclosed filovirus GP peptide (e.g., Zaire ebolavirus GPpeptide) include conventional non-amplifying mRNA immunization (see,e.g., Petsch et al., “Protective efficacy of in vitro synthesized,specific mRNA vaccines against influenza A virus infection,” Naturebiotechnology, 30(12):1210-6, 2012) and self-amplifying mRNAimmunization (see, e.g., Geall et al., “Nonviral delivery ofself-amplifying RNA vaccines,” PNAS, 109(36): 14604-14609, 2012; Maginiet al., “Self-Amplifying mRNA Vaccines Expressing Multiple ConservedInfluenza Antigens Confer Protection against Homologous andHeterosubtypic Viral Challenge,” PLoS One, 11(8):e0161193, 2016; andBrito et al., “Self-amplifying mRNA vaccines,” Adv Genet., 89:179-233,2015).

In some embodiments, administration of an effective amount of one ormore of the disclosed immunogens to a subject induces a neutralizing orprotective immune response in the subject. To assess neutralizationactivity, following immunization of a subject, serum can be collectedfrom the subject at appropriate time points, frozen, and stored forneutralization testing. Methods to assay for binding or neutralizationactivity are known to the person of ordinary skill in the art and arefurther described herein, and include, but are not limited to, ELISA,plaque reduction neutralization (PRNT) assays, microneutralizationassays, flow cytometry based assays, single-cycle infection assays. Insome embodiments, the serum neutralization activity can be assayed usinga panel of filovirus (e.g., Zaire ebolavirus) pseudoviruses.

VIII. Methods of Detection and Diagnosis

Methods are also provided for the detection of the presence ofantibodies to filovirus GP in a biological sample. The method can beused to identify a biological sample from a subject with a filovirus(such as ebolavirus) infection, or from a subject that had a priorinfection with a filovirus (such as an ebolavirus). In one example, thepresence of filovirus (for example, Zaire ebolavirus) GP is detected ina biological sample from a subject, and can be used to identify asubject with filovirus infection. The sample can be any sample form asubject that contains antibodies induced by the filovirus infection,including, but not limited to body fluids, such as blood, serum, plasma,sputum, spinal fluid or urine. The method of detection can includecontacting the sample with an isolated filovirus peptide as disclosedherein and under conditions sufficient to form an immune complex betweenthe peptide and the antibodies in the sample, and detecting the immunecomplex.

In some embodiments, the peptides disclosed herein are used to testvaccines. For example to test if a test vaccine elicits an immuneresponse that targets a particular antigenic site on the filovirus GP,such as antigenic site VI of Zaire ebolavirus as disclosed herein. Suchmethods involve immunizing a subject with a vaccine, and then screeninga sample from the subject that contains antibodies induced by theimmunization for antibody binding to the appropriate peptide.

Thus, the peptides disclosed herein can be used for serodiagnosis aswell as development of assays for evaluation of vaccine or therapeuticsor countermeasures or effectivity of these approaches.

VIII. Additional Description of Certain Embodiments

The present disclosure provides an immunogenic composition for inducingan immune response. The present disclosure also relates to prophylacticuses of the immunogenic compositions, for use in eliciting antibodies tothe immunogen. In certain embodiments, the present disclosure relates toimmunogenic compositions that provide a method of treating orsuppressing a Filovirus disease in a patient, the method comprisingadministering to a patient the immunogenic composition of thedisclosure.

In certain embodiments, the present disclosure relates to an immunogeniccomposition comprising one or more peptide fragments of a filovirusprotein for use in eliciting an immunogenic response in a mammal. Inother embodiments, the immunogenic composition can further comprisecarrier proteins conjugated to the peptide fragments. The immunogeniccomposition can further comprise an adjuvant.

In certain embodiments, the peptide fragment or peptide fragmentscomprise one or more of the amino acid sequences selected from thefollowing group of amino acid sequences or variants thereof:

SEQ ID NO: 1:  TTEDHKIMASENSSAMVQVHSQGREAAVSH SEQ ID NO: 2: ETAGNNNTHHQDTGEESASSGKLGLITN SEQ ID NO: 3: TGEESASSGKLGLITNTIAGVAGLITGGRR SEQ ID NO: 4: KNITDKIDQIIHDFVDKTLPDQGDNDNWWT SEQ ID NO: 5:  FVDKTLPDQGDNDNWWTSEQ ID NO: 6  (FX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWWT) wherein: X₂ is selected from V and I; X₃ from K and N; X₄ from T, P, and N; X₅ and X₇ from D and N; X₆ from G, T, D, and N; X₈ from N, D, and G; and X₉ from D andS. SEQ ID NO: 7  (KNITDKIXIQIIHDFX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NVVWT)wherein: X₂ is selected from V and I; X₃ from K and N; X₄ from T, P, and N; X₁, X₅ and X₇ from D and N; X₆ from G, T, D, and N; X₈ from N, D, and G; and X₉ from D and S. SEQ ID NO: 8 (TX₁₀EDHKIMASENSSAMVQVHSQGRX₁₁AAVSH) wherein: X₁₀ is selected from T and N; and X₁₁  from E and K.

In certain embodiments, a non-limiting acceptable carrier moleculeincludes keyhole limpet hemocyanin (KLH), DNA vectors, lentiviralvectors, nanoparticles, vesicular stomatitis virus (VSV), bovine serumalbumin, ovalbumin, fowl immunoglobulin, and cytosine-phosphate-guanine(CpG) oligodeoxynucleotides.

Clause 1. An immunogenic composition comprising a peptide fragmentcomprising an amino acid sequence of SEQ ID NO: 6(FX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWWT) wherein: X₂ is selected from V and I; X₃from K and N; X₄ from T, P, and N; X₅ and X₇ from D and N; X₆ from G, T,D, and N; X₈ from N, D, and G; and X₉ from D and S.

Clause 2. The immunogenic composition of Clause 1 wherein the peptidefragment comprises the amino acid sequence SEQ ID NO:5(FVDKTLPDQGDNDNWWT).

Clause 3. An immunogenic composition comprising a peptide fragmentcomprising an amino acid sequence of SEQ ID NO: 7(KNITDKIX₁QIIHDFX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWWT) wherein: X2 is selected fromV and I; X₃ from K and N; X₄ from T, P, and N; X₁, X₅ and X₇ from D andN; X₆ from G, T, D, and N; X₈ from N, D, and G; and X₉ from D and S.

Clause 4. The immunogenic composition of Clause 3 wherein the peptidefragment comprises the amino acid sequence SEQ ID NO: 4(KNITDKIDQIIHDFVDKTLPDQGDNDNWWT).

Clause 5. An immunogenic composition comprising a peptide fragmentcomprises an amino acid sequence of SEQ ID NO: 8(TX₁₀EDHKIMASENSSAMVQVHSQGRX₁₁AAVSH) wherein: X₁₀ is selected from T andN; and X₁₁ from E and K.

Clause 6. The immunogenic composition of Clause 5 wherein the peptidefragment comprises the amino acid sequence SEQ ID NO: 1(TTEDHKIMASENSSAMVQVHSQGREAAVSH).

Clause 7. An immunogenic composition comprising a peptide fragmentcomprising an amino acid sequence of SEQ ID NO: 2(ETAGNNNTHHQDTGEESASSGKLGLITN).

Clause 8. An immunogenic composition comprising a peptide fragmentcomprising an amino acid sequence of SEQ ID NO: 3(TGEESASSGKLGLITNTIAGVAGLITGGRR).

Clause 9. An immunogenic composition comprising a peptide fragmentcomprising an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 and apeptide fragment or peptide fragments selected from the list of peptidefragments encoded by amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 8.

Clause 10. An immunogenic composition comprising a peptide fragmenthaving amino acid sequence SEQ ID NO: 4 or SEQ ID NO: 7 and a peptidefragment or peptide fragments selected from the list of peptidefragments encoded by amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 8.

Clause 11. An immunogenic composition comprising a peptide fragment orpeptide fragments selected from the list of peptide fragments encoded bythe amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

Clause 12. The immunogenic composition of Clauses 1-11 furthercomprising an adjuvant.

Clause 13. The immunogenic composition of Clause 12 where in theadjuvant is oil in water, CpG, or a carrier protein.

Clause 14. The immunogenic composition of Clauses 1-11 furthercomprising a carrier protein whereby the carrier protein is conjugatedto each peptide fragment.

Clause 15. The immunogenic for composition of Clause 14 where in thecarrier protein is keyhole limpet hemocyanin.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Human Antibody Repertoire after VSV-Ebola VaccinationIdentifies Novel Targets and Virus-Neutralizing IgM Antibodies

Development of an effective vaccine against Ebola virus is of highpriority. However, knowledge about potential correlates of protectionand the durability of immune response after vaccination is limited.Here, we elucidate the human antibody repertoire after administration ofvesicular stomatitis virus (VSV)-Ebola vaccine at 3 million, 20 millionand 100 million plaque-forming units (PFU) and homologous VSV-Ebolavaccine boost in healthy adult volunteers. Whole genome-fragment phagedisplay libraries, expressing linear and conformational epitopes ofEbola glycoprotein (GP), showed higher diversity of antibody epitopes inindividuals vaccinated with 20 million PFU than in those vaccinated with3 million or 100 million PFU. Surface plasmon resonance kinetics showedhigher levels of GP-binding antibodies after a single vaccination with20 million or 100 million PFU than with 3 million PFU, and thesecorrelated strongly with neutralization titers. A second vaccination didnot boost antibody or virus neutralization titers, which declinedrapidly, and induced only minimal antibody affinity maturation. Isotypeanalysis revealed a predominant IgM response even after the secondvaccination, which contributed substantially to virus neutralization invitro. These findings may help identify new vaccine targets and aiddevelopment and evaluation of effective countermeasures against Ebola.

The recent 2014 epidemic of highly pathogenic Ebola virus (EBOV) inWestern Africa caused tens of thousands of infections and deaths. Withoccasional small outbreaks of new cases in Western Africa and thepossibility of long-term persistence of virus in some survivors, it isfeared that future outbreaks could occur and lead to severe epidemics.Therefore, development of an effective vaccine against Ebola is a highpriority, both for pre-epidemic preparedness and for rapid vaccinationto control future outbreaks. Protection against EBOV disease isattributed, at least partially, to the humoral immune response, aspassive transfer of antibodies to naive NHPs can protect recipientsagainst lethal EBOV challenge. Diverse ELISA, EBOV neutralization testsand immune parameters have been used to identify the vaccine correlatesof protection (Matassov, D. et al. J. Infect. Dis. 212 (Suppl. 2),S443-S451, 2015; Blaney, J. E. et al. PLoS Pathog. 9, e1003389, 2013;and Wong, G. et al. Sci. Transl. Med. 4, 158ra146, 2012). A recent studyalso investigated the role of T cells in EBOV-infected patients andfound that expression of the inhibitory molecules cytotoxic Tlymphocyte-associated protein 4 (CTLA-4) and programmed cell deathprotein 1 (PD-1) on CD4+ and CD8+ T cells was lower in survivors than infatal cases (Ruibal, P. et al. Nature 533, 100-104, 2016). However, todate, no single assay has been found to be predictive of protection, andthe correlation of antibody titers measured by various assays has notbeen clearly demonstrated. Coupled with the difficulty of conductingadequate randomized controlled trials to demonstrate vaccineeffectiveness on the basis of clinical EBOV disease, it is important toidentify and understand immune markers that are reasonably likely topredict clinical benefit and can facilitate evaluation of vaccinecandidates.

Recently, a recombinant VSV (rVSV)-based vaccine expressing Ebola Zairesurface glycoprotein from the Kikwit 1995 strain (rVSVΔG-ZEBOV-GP) wasreported to decrease transmission to close contacts in a ringvaccination study in Guinea (Henao-Restrepo, A. M. et al. Lancet 386,857-866, 2015). Here we performed an in-depth comprehensive analysis ofthe humoral immune response after primary rVSVΔG-ZEBOV-GP vaccinationadministered at 3 million, 20 million or 100 million PFU and homologousrVSVΔG-ZEBOV-GP vaccine boost in healthy US adult volunteers in a phase1 placebo-controlled trial (Regules, J. A. et al. N. Engl. J. Med.376(4):330-341, 2017, available online Apr. 1, 2015). Polyclonal serumwas analyzed quantitatively and qualitatively to elucidate antibodyepitope repertoires using gene-fragment phage display libraries (GFPDLs)and surface plasmon resonance (SPR) technology to measure realtimeantibody binding kinetics, antibody cross-reactivity, immunoglobulinisotypes, affinity maturation and antibody persistence in recipients ofthe rVSVΔG-ZEBOV-GP vaccine.

Results

Antibody Epitope Repertoire after Vaccination

A rVSVΔG-ZEBOV-GP vaccine was administered intramuscularly at 3 million,20 million or 100 million PFU to 39 healthy adult volunteers at the USNational Institute of Allergy and Infectious Diseases (NIAID). In eachdosing cohort, ten people received active vaccine, and three received asaline placebo. Serum samples from each individual were collected beforevaccination (day 0), after the first vaccination (day 28), after thesecond vaccination (days 42 and 56) and on days 84 and 180. To analyzethe epitope repertoire of serum samples from participants who receivedthe rVSVΔG-ZEBOV-GP vaccine, we generated a GFPDL containing 50- to1,000-bp fragments of the GP gene from the EBOV Mayinga strain (FIG. 9)or the homologous Kikwit strain (FIGS. 10A and 10B), with >107 uniquephage clones. These were expected to display all possible linear andconformational epitopes. The GP sequence of Mayinga of 1976 differs fromthe Kikwit strain of 1995 used in the vaccine by only nine amino acids,whereas the Makona strain of 2014 differs from Mayinga by 20 amino acids(FIGS. 10A and 10B).

Sequencing of the EBOV GFPDL confirmed a random distribution of size andsequence of inserts that spanned the entire GP (FIG. 11). To ascertainthat the antibody repertoire identified using the GFPDL approachrepresented both linear and conformational epitopes, we performed twoindependent experiments. First, a panel of EBOV-protective monoclonalantibodies (MAbs) was used to identify and confirm the potential of theEBOV GFPDL to map both linear and conformational antibody epitopes. Thispanel included 6D8 and 13F6, which are components of the MB-003 cocktailfor treatment of EBOV infection (FIG. 12), and conformation-dependentcross-reactive neutralizing and protective human MAbs derived from EBOVsurvivors (Flyak, A. I. et al. Cell 164, 392-405, 2016) (FIGS. 13A13C).The consensus epitope sequences obtained through GFPDL analysis weresimilar to the footprints previously identified for the MB-003 MAbs(Davidson, E. et al. J. Virol. 89, 10982-10992, 2015; Murin, C. D. etal. PNAS 111, 17182-17187, 2014) or for MAbs from survivors (Flyak, A.I. et al. Cell 164, 392-405, 2016), providing proof of concept that theGFPDL approach can identify linear and conformation-specific antibodiesin polyclonal serum after EBOV infection or vaccination.

Second, we determined the capacity of the EBOV GFPDL to adsorb EBOVGP-specific antibodies in post-vaccination polyclonal human sera. Aftertwo rounds of adsorption with the EBOV GFPDL, 85-92% of GP-specificantibodies in post-vaccination human sera were adsorbed by GP phagedisplay libraries, as determined by binding to EBOV GP in ELISA (FIG.14). Together, the epitope mapping of MAbs and adsorption studies inpost-vaccination polyclonal sera provided support for using the EBOV GPGFPDL to dissect the polyclonal antibody repertoires in human sera.

To study antibody responses after rVSVΔG-ZEBOV-GP vaccination, wecollected serum specimens from individuals vaccinated with 3 million, 20million or 100 million PFU vaccine or placebo control before vaccination(day 0), 28 d (day 28) after the primary vaccination and 28 d afterbooster vaccination (day 56). The sera of all participants in each dosegroup were pooled and used for mapping of overall antibody epitoperepertoires by EBOV GFPDL (FIG. 1).

Prevaccination sera and the placebo controls were bound by very fewphages. In the rVSVΔG-ZEBOV-GP-vaccinated groups, 4 weeks after thefirst vaccination, the number of bound phages was higher in sera fromthe 20-million-PFU dose (2.12×10⁶) group than in sera from the3-million-PFU and 100-million-PFU groups (1.12×10⁶ and 1.45×10⁶ phages,respectively) (FIG. 1a ). Sequencing of GP fragments expressed by phagesbound with sera after the first vaccination showed a high frequency ofbound phages displaying both small and large fragments mapping acrossthe N-terminal GP1 head domain and, to a lesser degree, the C-terminalGP2 stalk domain of the EBOV GP protein (FIG. 1b ). Sera from the3-million-PFU and 20-million-PFU cohorts contained antibodies thatmapped to the glycan cap and mucin domain and that recognized an epitopein the transmembrane region of the GP2 protein that was not captured bysera from the 100-million-PFU dose cohort. Sera from the 20-million-PFUdosing cohort additionally recognized several small and largeimmunodominant epitopes in the N-terminal half of EBOV GP mapping to thereceptor-binding region (RBR) and between the RBR and the glycan capdomain (FIG. 1b ).

Unexpectedly, after the second vaccination, the number of EBOV GFPDLbound phages decreased 2-10 times relatively to the first vaccination(FIG. 1a ). The antibody epitope profiles did not substantially change,apart from an apparent reduction in antibodies mapping to the GP2transmembrane region in the 3-million- and 20-million-PFU doserecipients, an increase in antibodies specific to the glycan cap in the20-million-PFU cohort, and a small reduction in antibodies recognizinglarge sequences in the mucin-like domain from the 100-million-PFU group(FIG. 1c ).

Antigenic Sites within EBOV GP

EBOV-neutralizing and/or protective MAbs, such as KZ52 and the MAbcocktails ZMAb, ZMapp and MB-003, have been shown to recognize epitopeswithin or flanking the mucin-like domain, glycan cap or base of GP(Davidson, E. et al. J. Virol. 89, 10982-10992, 2015; Murin, C. D. etal. PNAS 111, 17182-17187, 2014) (FIG. 2a ). Vaccination withrVSVΔG-ZEBOV-GP generated an immune response to 19 unique antigenicsites defined by six large antigenic regions (herein referred to as GP-Ithrough GP-VI) and 13 smaller antigenic sites (GP-II.1 through GP-V.7)contained within EBOV GP (FIG. 2a and FIGS. 6A and 6B). These antigenicregions and sites include several novel linear and conformationalepitopes, including GP-II, GP-II.1, GP-II.2, GP-IV.1, GP-IV.3, GP-V.1,GP-V.4, GP-V.5, GP-V.6, GP-V.7 and an immunodominant sequence (GP-VI) inthe transmembrane or cytoplasmic tail. The frequency of phagesexpressing these GP antigenic sites selected by sera after the first(FIG. 2b ) and second (FIG. 2c ) vaccinations for the threerVSVΔG-ZEBOV-GP dose groups are shown in FIGS. 6A and 6B. Antibodies inpost-vaccination sera from the 20-million-PFU dose group showed thehighest epitope diversity, as assessed by selecting phage clones frommost of the antigenic sites within GP, whereas the sera from the lower(3 million PFU) and higher (100 million PFU) dose groups containedantibodies that predominantly mapped to the C-terminal half of the EBOVGP. The surface exposure of each of these antigenic sites on the EBOV GPcrystal structure (Lee, J. E. et al. Nature 454, 177-182, 2008) (PDB3CSY; includes GP residues 33-189, 214-278, 299-310 and 502-599) and themodel of complete EBOV GP monomer (Yang, J. et al. Nat. Methods 12, 7-8,2015) (FIG. 15) are shown in FIG. 3. The surface representation showedthat most of the key antibody targets of rVSVΔG-ZEBOV-GP, includingseveral of the GP epitopes (GP-II, GP-II.1, GP-II.2, GP-IV.1, GP-IV.3,GP-V.1, GP-V.4, GP-V.5, GP-V.6, GP-V.7) discovered in this study, areexposed on the native ZEBOV GP structures. Analysis of sequence homologyof GP showed that some sites, including GP-II, GP-II.1, GP-IV.1, GP-IV.2and GP-VI, are >70% conserved between diverse EBOV strains, such asSudan, Bundibugyo, and Kikwit (FIG. 7). These data suggest thatantibodies induced after rVSVΔG-ZEBOV-GP vaccination against someconserved antigenic sites may cross-react with diverse EBOV strains,even though cross-protection has not been observed inrVSVΔG-ZEBOVGP-vaccinated NHPs challenged with EBOV Sudan virus29.

Correlation of GP Antibody Binding with EBOV Neutralization

Because the GFPDL analyses were carried out on the pooled serum samplesfrom each group, we performed quantitative and qualitative analyses ofindividual pre- and post-vaccination sera with recombinant glycosylatedGP produced in a mammalian system using an SPR-based real-time kineticsassay.

Binding kinetics of individual serum samples from the three vaccine-dosecohorts at early post-vaccination time points (28 d after firstvaccination and 14 and 28 d after second vaccination) and at later timepoints (day 84 and day 180) was performed using the GP from Mayinga andMakona strains (FIG. 4a,b ). Antibody binding titers of individual serumsamples (N=10 for each vaccine group at each time point) against GP weremeasured as resonance units (RU) in SPR (FIG. 4). Control sera fromplacebo did not show significant antibody binding to GP before or aftermock vaccination. After the first rVSVΔG-ZEBOV-GP vaccination (day 28),all samples reacted strongly with GP from Mayinga (FIG. 4a ) and Makonastrains (FIG. 4b ), with sera from the 20-million- and 100-million-PFUgroups showing higher binding (mean RU=2,152 and 2,056, respectively)than those of the 3-million-PFU group (mean RU=1,197), but thedifference among groups did not reach statistical significance. However,the mean serum antibody reactivity decreased marginally (mean RU=800 for3 million PFU; 1,608 for 20 million PFU; 1,715 for 100 million PFU) forall the vaccine dose groups by day 42 (14 d after the secondvaccination) and even further by day 56 (28 d after administration ofthe homologous boost; mean RU=679 for 3 million PFU; 1,409 for 20million PFU; 1,536 for 100 million PFU). By days 84 and 180, serumanti-GP titers had diminished substantially, such that by day 180, 80%of individuals had very weak GP-binding antibody levels for all vaccinegroups, though binding-antibody titers were marginally higher for thegroup that received the highest vaccine dose (mean GP binding RU=133 for3 million PFU; 260 for 20 million PFU; 490 for 100 million PFU). Weobserved a strong correlation between in vitro EBOV neutralizationtiters (FIG. 4c ) and the titers of serum GP-binding antibody, asmeasured by SPR, after rVSVΔG-ZEBOV-GP vaccination (r=0.75; P<0.0001)(FIG. 4d ). This analysis revealed that the replicating rVSVΔG-ZEBOV-GPvaccination generated strong GP-binding antibodies that peaked after thefirst vaccination but were not boosted after the second vaccination andwere not long lasting.

Antibody Affinity Maturation after Vaccination

To further investigate whether different rVSVΔG-ZEBOV-GP vaccine dosespromote anti-GP affinity maturation, we determined the dissociationrates (Kd) of post-vaccination serum antibody-antigen complexes usingSPR. Dissociation rate is independent of antibody concentration andprovides a measurement of overall affinity of polyclonal antibodybinding, as previously described (Khurana, S. et al. Sci. Transl. Med.3, 85ra48, 2011). The off-rates for polyclonal serum antibodies bound toGP were lower (indicating stronger affinity) at 14 d and 28 d after thesecond vaccination than at 28 d after the first vaccination, but thisdifference reached statistical significance only for the 20-million-PFUand 100-million-PFU groups (FIG. 4c ). However, polyclonal antibodyoff-rates (Kd=10⁻²-10⁻³/s) were low, even after two vaccinations,compared to some other human viral vaccines (Khurana, S. et al. Sci.Transl. Med. 3, 85ra48, 2011). GP-specific antibody off-rates after thefirst (day 28) and second vaccination (day 56) correlated strongly withvirus neutralization titers (r=−0.876, P=<0.0001), emphasizing thepotential importance of antibody affinity maturation for antiviralactivity (FIG. 4f ). These observations suggest that the higherrVSVΔG-ZEBOV-GP vaccine doses (20 million PFU and 100 million PFU)promote better antibody affinity maturation to GP than the lower vaccinedose.

Anti-GP Isotype and EBOV-Neutralizing Capacity

Isotype analysis of the GP-binding antibodies demonstratedrepresentation of all isotypes (IgA, IgG and IgM) and IgG subclasses inpostvaccination sera (FIG. 5a ). We were surprised to find that most ofthe GP-binding antibodies both after primary and after boostervaccinations, were of IgM isotype in all vaccine-dose groups (FIG. 5a ).The second most abundant GP-binding antibodies were of IgA isotype, andtheir frequency in serum increased with dosage (mean IgA=4% for 3million PFU; 10% for 20 million PFU; 20% for 100 million PFU) at day 56.The anti-GP isotype reactivity was also analyzed by ELISA (FIGS.16A-16C). Antibody isotyping of GP-binding antibodies inpost-vaccination sera by ELISA showed concordance with the isotypesdetermined by SPR, but ELISA underestimated the proportion of GP-bindingIgM antibodies compared to SPR. In contrast, at later time points (day84 and day 180) after vaccination, the GP-binding antibodies remainingin the sera (FIG. 4a ) were of IgA or IgG subclasses (FIG. 5a ),suggesting that most of the anti-GP antibody response generated earlyafter vaccination is of the IgM isotype, which does not provide along-lasting systemic anti-GP antibody response. After vaccination, ofthe total GP-bound IgG antibodies, IgG1, IgG2 and IgG3 contributed mostto GP binding in the 3-million-PFU and 20-million-PFU vaccine cohort,whereas for the 100-million-PFU dose a significant amount of anti-GPantibodies were of IgG3 and IgG4 subclasses (FIG. 17).

To understand the functional role of IgM antibodies in thepostvaccination response, we evaluated their contribution to virusneutralization and compared them to IgG antibodies in post-vaccinationsera. We used anti-human IgG and anti-human IgM affinity chromatographycolumns to purify IgG and IgM antibodies from three sera samples, takenafter the second vaccination, that showed similar amounts of GP-bindingIgG and IgM antibodies and evaluated binding to the GP in SPR (FIG. 5b), isotype specificity (FIG. 5c,d ) and virus neutralization of Kikwitand Makona EBOV strains (FIG. 5e,f ). The IgG/IgM ratio determined fromaffinity chromatography in post-vaccination sera was in good agreementwith that determined by SPR. The purified IgG and IgM antibodies fromall sera reacted with GP, and, as expected, the IgG antibodies showedhigher affinity (slower dissociation) to GP than IgM in SPR (FIG. 5b ).The purity of antibody isotypes was confirmed by human IgG- and humanIgM-specific secondary antibodies using SPR (FIG. 5c,d ). The purifiedpost-vaccination IgM antibodies at serum concentration levelscontributed 40-50% to virus neutralization, and these results weresimilar to those with IgG antibodies purified from post-vaccination serafrom the same individuals (FIGS. 5E and 5F). These results suggest thatanti-GP IgM antibodies could have an important role in protectionagainst EBOV disease in vivo.

The results of our study demonstrate independent evolution of antibodybinding patterns to EBOV GP—in terms of epitope repertoire diversity,affinity maturation and isotype switching—in the three vaccine-dosegroups after the first and second vaccinations and show an importantcontribution of anti-GP IgM antibodies to EBOV neutralization.

Discussion

hi-depth understanding of the humoral immune response to Ebola vaccinesunder advanced development is required to identify meaningful correlatesof protection in humans and animal models to facilitate evaluation ofeffective vaccine candidates. The epitope-binding patterns in the GPantigenic sites were most diverse in the 20-million-PFU dose samples.This effect, in which a more diverse antibody repertoire is generatedfrom a lower vaccine dose, has been observed in multiple human influenzavaccination studies and was linked with optimal CD4+ T cell help, whichmay affect the B cell and T cell response differently after vaccination(Chung, K. Y. et al. Vaccine 33, 3953-3962, 2015; Nicholson, K. G. etal. Lancet 357, 1937-1943, 2001; Jackson, L. A. et al. J. Am. Med.Assoc. 314, 237-246, 2015; and Mulligan, M. J. et al. J. Am. Med. Assoc.312, 1409-1419, 2014). The lower numbers of captured GFPDL phages afterthe second vaccination, as compared to the first vaccination, suggestthat pre-existing antibodies against rVSVΔG-ZEBOV-GP after the firstvaccination may have impeded replication of the rVSV vector and possiblymasked some GP epitopes. Notably, the 20-million-PFU dose, selected forthe phase ⅔ clinical trials in Western Africa on the basis of safety andin vitro neutralization data, generated the broadest antibody repertoireafter vaccination.

After finding that a substantial proportion of anti-GP antibodies wereof IgM and IgA isotypes, we performed GFPDL analysis to determinespecific epitopes recognized by IgA, IgG and IgM antibodies inpost-vaccination sera. In the sera pooled from each group after thefirst vaccination, the number of bound phages was approximately twofoldhigher in IgM-bound antibodies than in protein A/G-bound (primarily IgG)antibodies, and in IgA-bound antibodies was about tenfold lower for allvaccine groups (FIG. 8). We performed an additional analysis of serafrom individuals in the 20-million-PFU and 100-million-PFU groups usingIgA-, IgG- and IgM-specific capture beads to further define the fineepitope specificity of these antibodies after the first vaccinationusing homologous Kikwit GP EBOV strain GFPDL (FIGS. 18A-18C). Theepitope repertoires of IgG-specific antibodies in individualpost-vaccination sera were similar to those identified in IgG antibodiesfrom pooled post-vaccination sera from the 20-million-PFU and100-million-PFU dose groups (FIG. 1). However, the IgM antibody epitoperepertoire in the 100-million-PFU dose group was more diverse than thatof the 20-million-PFU group, which predominantly recognized themucin-like domain. The individual IgM GFPDL responses quantitativelytracked the SPR data for total GP-binding antibodies, which measuresbinding of all anti-GP antibody isotypes. In both dose groups (20million and 100 million PFU), IgA-specific polyclonal repertoire wasmore focused on the glycan cap and mucinlike domain, but sera from the20-million-PFU group recognized the antigenic site V.7 at the C terminusof GP1 with higher frequency (FIGS. 18A-18C). One possible limitation ofGFPDL-based assessment is that it is unlikely to detect paratopicinteractions that require post-translational modification or rarequaternary epitopes that cross GP protomers. However, 85-92% of anti-GPantibodies from post-vaccination sera were removed by adsorption withthe EBOV GP GFPDL, supporting the use of the EBOV GP GFPDL for analysesof human sera, as has been observed with other viral antigens, includingdifferent influenza strains, respiratory syncytial virus (RSV) protein F(RSV-F) and heavily glycosylated RSV-G. Moreover, binding to properlyfolded glycosylated Ebola GP in SPR can overcome these limitations andprovide additional insight into the post-vaccination anti-GP polyclonalantibody response. Real-time antibody kinetics of individualpost-vaccination sera by SPR revealed several unexpected findings,including lack of impact of the second vaccination on anti-GP responses,a fast decay of anti-GP titers within 2 months after the secondvaccination, limited antibody class switching and modest antibodyaffinity maturation.

The observation of low antibody class switching after rVSVΔGZEBOV-GPvaccination is notable. Our findings suggest that IgM antibodies are thepredominant isotype and decayed rapidly after the first and secondvaccination. Although IgM antibodies are of low affinity, theirmultivalency compensates for overall binding avidity and helps in virusneutralization. Therefore, IgM antibodies may contribute to protectionagainst infection, which would explain the relatively rapid protectionthat has been described in NHP studies and the rVSVΔGZEBOV-GP ringvaccination study in Africa. The amount of IgA in most post-vaccinationsera samples was too low to purify and perform a reproducible EBOVneutralization assay. IgA purified from sera from two vaccine recipientsshowed that GP-specific IgA antibodies can neutralize virus in vitro butto a lesser extent than IgM antibodies.

Most ELISAs used for evaluation of Ebola vaccines measure predominantlyanti-GP IgG antibody titers, because they rely on anti-human IgGsecondary antibodies. Such ELISA titers alone may underestimate the fullspectrum of the vaccine-induced immune response. Although incorporatingan anti-human IgM secondary antibody in the ELISA may help mitigate thisdeficiency, the washing steps involved in the ELISA process, which areimportant to reduce nonspecific binding, may elute most of thelow-affinity IgM antibodies. In addition, anti-GP IgA antibodies maycontribute to protection against EBOV infection and disease in vivo,especially at mucosal surfaces. In contrast, the SPR approach capturesall antibody classes, including IgM, IgA and IgG, and is also moreappropriate for maintaining the native structure of EBOV GP andpreserving conformational epitopes.

Class switching and antibody affinity maturation require continuoussignals from T cells in the form of cytokines and comigration of antigenspecific follicular helper T cells and B cells into germinal centers inlymph nodes. The influence of pre-existing rVSVΔG-ZEBOV-GP-specificantibodies or anti-VSV vector responses at the time of the boostervaccination, with their attenuating effects on rVSV replication andmasking of GP epitopes, could adversely affect the formation of germinalcenters and prevent antibody class switching, affinity maturation anddurable response, as observed in previous vaccine studies. A secondvaccination or prime-boost with alternative vaccine platforms (includingdifferent VSV serotype vectors) could provide a meaningful increase inaffinity maturation and a better immune response, as was observed inprime-boost H5N1 and H7 influenza vaccine studies in humans. In thesestudies, it was observed that a 3-month minimum interval between thefirst and second vaccine doses was required for optimalneutralizing-antibody response and antibody affinity maturation.

In summary, we have demonstrated independent evolution of antibodyimmune responses—in terms of antibody epitope repertoire diversity,affinity maturation, durability and isotype switching—after vaccinationwith a live rVSV vector-based vaccine in three vaccine-dose groups andrevealed the importance of a predominantly anti-GP IgM response for EBOVneutralization. These findings could have significant implications forfurther development and evaluation of Ebola vaccines. Future Ebolavaccine studies should follow the rate of decay of anti-VSV antibodiesto identify the time interval needed for a booster vaccination togenerate optimal antibody affinity maturation and durable antibodyresponses. Our observations suggest that it is important to developappropriate assays that can provide in-depth understanding ofpost-vaccination and postinfection antibody responses to help guidedevelopment and evaluation of effective Ebola countermeasures such astherapeutics and vaccines.

Methods

Sera Samples and Monoclonal Antibodies.

Monoclonal antibodies (MAbs) and recombinant EBOV GP used in this studywere purchased from IBT Bioservices Inc. Cross-reactiveconformation-dependent neutralizing and protective human MAb 289 and MAb324 from EBOV survivors were obtained from J. Crowe24. Phase 1,double-blind, placebo-controlled, dose-escalation trials with staggeredenrollment were designed across three dose levels as outlined in Reguleset al. (N. Engl. J. Med. 376(4):330-341, 2017, available online Apr. 1,2015). Briefly, the rVSVΔG-ZEBOV-GP vaccine consisting of the rVSVstrain Indiana and the glycoprotein of the EBOV Kikwit 1995 strainreplacing the gene encoding the VSV envelope glycoprotein wasadministered at 3 million, 20 million or 100 million PFU in the form ofa 1-mL injection in the deltoid muscle of healthy adult men and womenaccording to protocols approved by the institutional review board at theUS National Institutes of Health NIAID site. Written informed consentwas obtained from all the volunteers before enrollment. Within eachdosing cohort, 10 received active vaccine and 3 received a salineplacebo. Serum samples from each individual were collected beforevaccination (day 0), after the first vaccination (day 28), after thesecond vaccination (days 42 and 56), and on day 84 and day 180(ClinicalTrials.gov number NCT02280408). Samples were anonymous, andpermission to test these deidentified samples in different antibodyassays was obtained from the US Food and Drug Administration's ResearchInvolving Human Subjects Committee (FDA-RIHSC) under exemption protocol#15-0B; all assays done fell within the permissible usages in theoriginal consent.

PsVN Assay.

Pseudovirion neutralization assay (PsVNA) against the homologousZaire-Kikwit strain glycoprotein was performed as described previously(Regules, J. A. et al. N. Engl. J. Med. 376(4):330-341, 2017, availableonline Apr. 1, 2015).

GFPDL Construction.

cDNAs complementary to the envelope glycoprotein-encoding gene of EBOVMayinga or Kikwit strain were chemically synthesized and used forcloning. A gIII display-based phage vector, fSK-9-3, where the desiredpolypeptide can be displayed on the surface of the phage as agIII-fusion protein, was used. Purified DNA containing Ebola GP wasdigested with DNase I to obtain gene fragments of 50-1,000 bp and usedfor GFPDL construction as described previously (Khurana, S. et al. PLoSMed. 6, e1000049, 2009; Khurana, S. et al. Sci. Transl. Med. 3, 85ra48,2011). As the phage libraries were constructed from the whole gene, theypotentially display all possible known or unknown viral protein segmentsranging in size from 15 to 350 amino acids as fusion proteins on thesurface of the bacteriophage.

Adsorption of Polyclonal Human Sera on EBOV GFPDL Phages and ResidualReactivity to EBOV GP.

Prior to panning of GFPDL, 500 □l tenfold-diluted serum antibodies frompost-vaccination pooled human sera (n=10 each from the 20-million-PFUand 100-million-PFU groups; 5 μl serum from each vaccine to obtain atenfold dilution for the pooled sera) were adsorbed by incubation inEBOV GFPDL phage-coated petri dishes. To ascertain residual antibodyspecificity, an ELISA was performed in wells coated with 200 ng/100 μlrecombinant EBOV GP. After blocking with 20 mM PBS, pH 7.4, containing0.05% Tween-20 (PBST) containing 2% milk, serial dilutions of humanserum (with or without adsorption) in blocking solution were added toeach well and incubated for 1 h at room temperature (RT) before additionof 5,000-fold diluted HRP-conjugated goat anti-human IgA IgG IgMantibody and developed by 100 μl o-phenylenediamine dihydrochloride(OPD) substrate solution. Absorbance was measured at 490 nm.

Affinity Selection of EBOV GP GFPDL Phages with rVSVΔG-ZEBOV-GPPost-Vaccination Polyclonal Human Sera.

Prior to panning of GFPDL with polyclonal serum antibodies, serumcomponents that could nonspecifically interact with phage proteins wereremoved by incubation in UV-killed M13K07 phage-coated petri dishes.Equal volumes of pooled polyclonal human sera from each cohort were usedfor each round of GFPDL panning. All samples in each group (N=10) werepooled for GFPDL analysis. Subsequent GFPDL affinity selection wascarried out in solution (with protein A/G) as previously described(Khurana, S. et al. PLoS Med. 6, e1000049, 2009; Khurana, S. et al. Sci.TransL Med. 3, 85ra48, 2011). GFPDL affinity selection experiments wereperformed in quadruplicate (two independent experiments by two differentinvestigators, who were blinded to sample identity) and showed similarnumbers of phage clones and epitope repertoires. Additional antibodyepitope repertoire analysis was performed using individualpost-vaccination sera with similar neutralization titers from20-million- and 100-million-PFU dose groups using IgA-, IgG- andIgM-specific capture beads to further define the fine epitopespecificity of these antibodies in the individual sera using EBOV KikwitGP GFPDL. A model for the complete Zaire strain GP generated usingI-TASSER (Yang, J. et al. Nat. Methods 12, 7-8, 2015) was used torepresent the antigenic sites on the structure. The crystal structure ofEbola GP (PDB 3CSY) was used as a reference (Lee, J. E. et al. Nature454, 177-182, 2008).

Binding of rVSVΔG-ZEBOV-GP-Vaccinated Human Sera to Recombinant GP andOff-Rate Measurements by Surface Plasmon Resonance (SPR).

Steadystate equilibrium binding of pre- and post-vaccination humanpolyclonal sera from every individual in the study was monitored at 25°C. using a ProteOn surface plasmon resonance (Bio-Rad). The purifiedrecombinant GP was coupled to a GLC sensor chip via amine coupling witheither 100 or 500 resonance units (RU) in the test flow channels. Theprotein density on the chip was optimized to measure only monovalentinteractions independent of the antibody isotype. Samples of 300 μlfreshly prepared sera at tenfold and 100-fold dilution BSAPBST buffer(PBS, pH 7.4, with Tween-20 and BSA) were injected at a flow rate of 50□l/min (240 s contact duration) for association, and disassociation wasperformed over a 1,200-s interval. Responses from the protein surfacewere corrected for the response from a mock surface and for responsesfrom a buffer-only injection. SPR was performed with serially dilutedsera (tenfold and 100-fold dilutions) of each sample in this study suchthat the SPR signal of the samples between 5 to 100 RU was used forfurther quantitative analysis. The maximum resonance units (max RU) datawere calculated by multiplying the observed RU signal with the dilutionfactor for each serum sample to provide the data for an undiluted serumsample. Antibody isotype analysis for the GP-bound antibodies inpost-vaccination polyclonal sera was performed using SPR. Total antibodybinding and isotype analysis were calculated with Bio-Rad ProteOnmanager software (version 3.0.1). All SPR experiments were performedtwice, and the researchers performing the assay were blinded to sampleidentity. In these optimized SPR conditions, the variation for eachsample in duplicate SPR runs was <6%. Antibody off-rate constants, whichdescribe the stability of the complex, i.e., the fraction of complexesdecaying per second, were determined directly from thepost-rVSVΔG-ZEBOV-GP-vaccination human polyclonal sera sampleinteraction with rGP protein using SPR (as described above) andcalculated using the Bio-Rad ProteOn manager software for theheterogeneous sample model.

Purification of IgG and IgM Antibodies from the Post-Vaccination Sera.

Fivefold-diluted post-second-vaccination sera were added to anti-humanIgG or anti-human IgM immune-affinity resin and incubated for 1 h at RTon an end-to-end shaker before washing and purification of boundantibodies. The antibodies were eluted by 4 M magnesium chloride in 10mM Tris (pH 7), followed by desalting. The purified IgG and IgMantibodies were normalized by volume to original serum concentrationsand tested for GP binding then isotyped using anti-human IgG andanti-human IgM secondary antibodies in SPR to confirm the purity of eachantibody isotype preparation. The purified IgG and IgM antibodies weresubjected to virus microneutralization assay.

Statistical Analyses.

The statistical significance of group differences was determined byordinary one-way ANOVA and Bonferroni's multiple-comparisons method.P<0.05 was considered significant with a 95% confidence interval.Correlations were calculated with the Pearson method, and P values forcorrelation were calculated by two-tailed test.

Data Availability.

The data sets generated during and/or analyzed during the study areavailable from the corresponding author upon reasonable request.

Example 2

This example demonstrates that immunization of rabbits and mice withselected peptide fragments conjugated to the carrier protein, keyholelimpet hemocyanin (KLH), generates strong binding antibodies against thematched Zaire ebolavirus and protects against ebolavirus infection.

An alignment of the glycoprotein (GP) amino acid sequences of 6 speciesof ebolavirus (Mayinga (1976), Kikwit (1995), Makona (2014), Bundibugyo(2012), Sudan (2000), Tai Forest) and Marburg margburgvirus is providedin FIGS. 19A-19C. Antigenic sites of ebolavirus GP as described hereinare reflected in the table shown in FIG. 20, which shows the sequence ofthe antigenic site sin the context of the Zaire ebolavirus Mayinga GPsequence set forth as SEQ ID NO: 35.

Peptide Fragment Conjugation to KLH Carrier Protein:

The conjugation procedure followed for peptide conjugation to MaleimideActivated mcKLH was performed as described in the product manual ofImject® Maleimide Activated mcKLH (Product 77605, Thermo Scientific).Several different fragments of SEQ ID NO: 35 were conjugated to themcKLH.

Rabbit Immunization Studies:

Female New Zealand white rabbits were immunized three timesintra-muscularly at 21-days interval with 25 micrograms of KLHconjugated peptides. Sera was collected before (pre-vaccination) andafter 3rd vaccination and analyzed for binding antibodies using SurfacePlasmon Resonance (SPR) and neutralization assay.

Binding of Glycoprotein (GP) Peptide Vaccinated Rabbit Sera to DifferentEBOV GP's and Cross-Reactivity Measurements by Surface Plasmon Resonance(SPR):

Steady-state equilibrium binding of pre- and post-KLH-GP vaccinatedrabbit polyclonal sera from every rabbit was monitored at 25 degreesCelsius using a ProteOn surface plasmon resonance (Bio Rad). Thepurified GP were coupled to a GLC sensor chip via amine coupling with500 resonance units (RU) in the test flow channels. The protein densityon the chip was optimized such as to measure only monovalentinteractions. Samples of 300 microliters of freshly prepared sera at10-fold dilution in BSA-PBST buffer (PBS pH 7.4 buffer with Tween-20 andBSA) were injected at a flow rate of 50 microliters/min (240 sec contactduration) for association, and disassociation was performed over a1200-second interval. Responses from the protein surface were correctedfor the response from a mock surface and for responses from abuffer-only injection. The resonance units (RU) data shown for each serain FIG. 21 was the observed RU signal for each serum sample.

Pseudovirion Neutralization (PsVN) Assay:

Pseudovirion neutralization assay (PsVNA) against the homologousZaire-Kikwit strain glycoprotein was performed as described previously(Regules, J. A. et al. N. Engl. J. Med. 376(4):330-341, 2017, availableonline Apr. 1, 2015)

Discussion:

Immunization of rabbits with selected ebolavirus antigenic peptidesgenerated strong binding antibodies against the matched Ebola virusstrain. Some of the immunized rabbit sera also showed strong crossreactivity to diverse ebolavirus strains including Bundibugyo and theMakona strain from the recent 2014 epidemic in Western Africa as well asdiverse Sudan virus.

Rabbit anti-GP peptide post-vaccination sera were analyzed for virusneutralization in the PsVNA against the Zaire Mayinga strain showed thatfive antigenic peptides generated neutralizing antibodies (anti-GP335-364, GP 457-484, GP 469-498, GP 617-645 and GP 630-646) against theMayinga (1976) strain. Two of these peptides from the carboxy terminusof GP (GP 617-645 and GP 630-646) upon rabbit immunization generatedstrong neutralizing titers when tested in the conventional BSL4 basedwild type Ebola virus with end-point titers of 320 and 640. Results areshown in the following table:

Neutralization titers of anti-GP peptide Rabbit sera GP 457-484 GP630-646 GP 335-364 GP 469-498 GP 617-645 Pseudovirus Neut- Mayinga 62.24194.1 85 44.86 79.14 Wild type PRNT- Mayinga 40 640 40.00 40 320Pseudovirus Neut- Kikwit 45 179 62 Pseudovirus Neut- Makona 38.74 84.1441.04

These anti-peptide rabbit sera also showed cross-neutralization of thedistinct Kikwit and 2014-Makona strain as well with C-terminal peptide(GP 630-646) immunized sera showing the highest cross-neutralizationtiters.

Additional immunization assays were performed as discussed above usingthe peptides shown in FIG. 22. Again, peptides from the C-terminus of GP(GP 282-305, GP 343-368 and GP 520-547) that elicited a neutralizingimmune response.

These rabbit studies confirmed that these antigenic GP peptides areimmunogenic when vaccinated into animals that bind diverse multipleebolaviruses and five of these conserved peptides generated neutralizingantibodies that also cross-neutralized diverse ebolaviruses.

Mouse Immunization and Challenge Studies

In another experiment (depicted in FIG. 23), female BALB/C mice wereimmunized on days 0 and 29 intra-muscularly with 20 micrograms of a KLHconjugated peptide (peptide fragments comprising SEQ ID NO: 17, SEQ IDNO: 21, SEQ ID NO: 3, SEQ ID NO: 29, SEQ ID NO: 10), each KLH-conjugatedpeptide fragment was screened separately. Furthermore, Female BALB/Cmice were also used to study immunization with a combination of peptidefragments, a total of 20 micrograms comprising 4 microgram of each ofthe five KLH conjugated peptides (peptide fragments comprising SEQ IDNO: 17, SEQ ID NO: 21, SEQ ID NO: 3, SEQ ID NO: 29, SEQ ID NO: 10), with10 mice per group). As a control, mice were injected with 20 microgramsof KLH only, or a particle based vaccine (VRP) expressing full lengthZaire EBOV GP.

At day 63, the mice were challenged with 100 pfu Zaire ebolavirus.Immunization with peptides from antigenic sites V.7 (SEQ ID NO: 3) andVI (SEQ IDNO: 10) induced sterilizing immunity to the viral challenge(FIG. 23), and protected against weight loss (FIG. 24).

Following immunization but prior to the viral challenge, sera wascollected to assess the specificity of the immune response by surfaceplasmon resonance. As shown in FIG. 25, the conserved antigenic sites inthe C-terminal region of GP1 and GP2 generate strong binding antibodiesto both Mayinga and Makona GP.

Characterization of Human Sera from Ebolavirus Survivors

Additional assays were performed to determine if sera from subjectspreviously infected with Zaire ebolavirus targeted peptidescorresponding to the antigenic sites identified herein (FIG. 26-27).Sera from infected human subjects targeted several of the identifiedsites, some of which strongly correlated with ebolavirus neutralizationtiters. The spatial structure of several of these sites is shown in FIG.28

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. An isolated peptide, comprising: (A) an amino acid sequence set forthas (antigenic site VI, 617-645): SEQ ID NO: 9:KNITDKIX₁QIIHDFX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWW, wherein X₁ is D or N, X₂is V orI, X₃ is K or N, X₄ is T, P, or N, X₅ is D or N, X₆ is G, T, D, or N, X₇is D or N, X₈ is N, D, or G, and X₉ is D or S; (B) an amino acidsequence set forth as (antigenic site VI.1, 630-645): SEQ ID NO: 6:FX₂DX₃X₄TLPXSQX₆X₇X₈X₉NWWT, wherein X₂ is V or I, X₃ is K or N, X₄ is T,P, or N, X₅ is D or N; X₆ is G, T, D, or N; X₇ is D or N, X₈ is N, D, orG; and X₉ is D or S; (C) an amino acid sequence set forth as (antigenicsite V.7, 469-498): SEQ ID NO: 3: TGEESASSGKLGLITNTIAGVAGLITGGRR, Zaireebolavirus Mayinga, Kikwit, Makona); SEQ ID NO: 32:NPIDISESTEPGLLTNTIRGVANLLTGSRR (Bundibugyo ebolavirus); SEQ ID NO: 33:TVLPQESTSNGLITSTVTGILGSLGLRKR (Sudan ebolavirus); SEQ ID NO: 34:RAVHPDELSGPGFLTNTIRGVTNLLTGSRR (Tai forest ebolavirus); or SEQ ID NO:55: GLINAPIDFDPVPNTKTIFDESSSSGASAE (Marburg marburgvirus); or (D) anamino acid sequence set forth as (antigenic site V.6, 457-484): SEQ IDNO: 2: ETAGNNNTHHQDTGEESASSGKLGLITN (Zaire ebolavirus Mayinga, Kikwit,Makona); SEQ ID NO: 26: MITSHDTDSNRPNPIDISESTEPGLLTN (Bundibugyoebolavirus); SEQ ID NO: 27: LTTPENITTAVKTVLPQESTSNGLITS (Sudanebolavirus); SEQ ID NO: 28: LPEQHTAASAIPRAVHPDELSGPGFLTN (Tai Forestebolavirus); or SEQ ID NO: 56: LWREGDMFPFLDGLINAPIDFDPVPTK (Marburgmarburgvirus); and wherein the peptide is no more than 100 amino acidsin length and induces a neutralizing immune response to filovirus in asubject.
 2. The isolated peptide of claim 1, wherein the peptidecomprises the antigenic site VI amino acid sequence set forth as any oneof: SEQ ID NO: 10: KNITDKIDQIIHDFVDKTLPDQGDNDNWW(Zaire ebolavirus Mayinga, Kikwit, Makona); SEQ ID NO: 11:KNITDKINQIIHDFIDKPLPDQTDNDNWW (Bundibugyo ebolavirus); SEQ ID NO: 12: KNITDKIDQIIHDFIDNPLPNQDNDDNWW (Sudan ebolavirus); SEQ ID NO: 13:KNITDKINQIIHDFVDNNLPNQNDGSNWW (Ta ï  Forest ebolavirus); andSEQ ID NO: 53: KNISEQIDQIKKDEQKEGTGWGLGGKWW (Marburg marburgvirus).


3. The isolated peptide of claim 1, wherein the peptide comprises theantigenic site VI amino acid sequence set forth as SEQ ID NO: 7:KNITDKIX₁QIIHDFX₂DX₃X₄TLPX₅QX₆X₇X₈X₉NWWT, wherein: X₁ is D or N, X₂ is Vand I, X₃ is K or N, X₄ is T, P, or N, X₅ is D or N, X₆ is G, T, D, orN, X₇ is D or N, X₈ is N, D, or G; and X₉ is D or S.
 4. The isolatedpeptide of claim 3, wherein the peptide comprises the antigenic site VIamino acid sequence set forth as any one of: SEQ ID NO: 4: KNITDKIDQIIHDFVDKTLPDQGDNDNWWT) (Zaire ebolavirus Mayinga, Kikwit, Makona);SEQ ID  NO: 14: KNITDKINQIIHDFIDKPLPDQTDNDNWWT (Bundibugyo ebolavirus);SEQ ID NO: 15:  KNITDKIDQIIHDFIDNPLPNQDNDDNWWT  (Sudan ebolavirus);SEQ ID NO: 16:  KNITDKINQIIHDFVDNNLPNQNDGSNWWT  (Taï Forest ebolavirus);  and SEQ ID NO: 54:  KNISEQIDQIKKDEQKEGTGWGLGGKWWT(Marburg marburgvirus).


5. The isolated peptide of claim 1, wherein the peptide comprises theantigenic site VI.1 amino acid sequence set forth as SEQ ID NO: 5 orresidues 14-30 of any one of SEQ ID NOs: 14-16 or
 54. 6. The isolatedpeptide of claim 1, wherein the peptide comprises the antigenic site V.6amino acid sequence set forth as any one of: SEQ ID NO: 43: SETAGNNNTHHQDTGEESASSGKLGLITN; SEQ ID NO: 44: TMITSHDTDSNRPNPIDISESTEPGLLTN; SEQ ID NO: 45: TLTTPENITTAVKTVLPQESTSNGLITS; SEQ ID NO: 46: TLPEQHTAASAIPRAVHPDELSGPGFLTN; and SEQ ID NO: 57:ILWREGDMFPFLDGLINAPIDFDPVPTK (Marburg marburgvirus).


7. The isolated peptide of claim 1, wherein the peptide consists of orconsists essentially of the amino acid sequence set forth as any one ofSEQ ID NOs: 2-5, 10-16, 26-28, 32-34, 43-46, or 53-57, or residues 14-30of any one of SEQ ID NOs: 14-16 or
 54. 8. An isolated peptide,comprising: (A) an amino acid sequence set forth as (antigenic siteIV.1, 282-305): SEQ ID NO: 17:  DTTIGEWAFWETKKNLTRKIRSEE(Zaire ebolavirus Mayinga, Kikwit, Makona); SEQ ID NO: 18: DTGVGEWAFWENKKNFTKTLSSEE (Bundibugyo ebolavirus); SEQ ID NO: 19: NADIGEWAFWENKKNLSEQLRGEE (Sudan ebolavirus); SEQ ID NO: 20: DTSMGEWAFWENKKNFKKTLSSEE (Ta ï Forest ebolavirus);  or SEQ ID NO: 58: DEDLATSGSGSGEREPHTTSD (Marburg marburgvirus).

(B) an amino acid sequence set forth as (antigenic site V.1, 343-368):SEQ ID NO: 21:  ASENSSAMVQVHSQGREAAVSHLTTL(Zaire ebolavirus Mayinga, Kikwit); SEQ ID NO: 22: ASENSSAMVQVHSQGRKAAVSHLTTL (Zaire ebolavirus Makona); SEQ ID NO: 23: VPKDPASVVQVRDLQRENTVPTSP (Bundibugyo ebolavirus); SEQ ID NO: 24: VPKNSPGVVPLHIPEGETTLPSQNST (Sudan ebolavirus);SEQ ID NO: 25: VSEDSTPVVQMQNIKGKDTMPTTV  (Ta ï Forest ebolavirus);  orSEQ ID NO: 59:  LDKNNTTAQPSMPPHNTTTISTNNTS (Marburg marburgvirus);

(C) an amino acid sequence set forth as (antigenic site V.10, 520-547):SEQ ID NO: 29:  TQDEGAAIGLAWIPYFGPAAEGIYIEGL (Zaire ebolavirus Mayinga);SEQ ID NO: 30:  TQDEGAAIGLAWIPYFGPAAEGIYTEGL(Zaire ebolavirus Kikwit, Makona, Sudan  ebolavirus); SEQ ID NO: 31: TQDEGAAIGLAWIPYFGPAAEGIYTEGI (Bundibugyo ebolavirus, Ta ï Forest ebolavirus); or SEQ ID NO: 60:  VQEDDLAAGLSWIPFFGPGIEGLYTAGL(Marburg marburgvirus);

(D) an amino acid sequence set forth as (antigenic site II.1, 152-220):SEQ ID NO: 47: AFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRY (Zaire ebolavirus Mayinga, Kikwit, Makona); SEQID NO: 48: AFHKEGAFFLYDRLASTIIYRSTTFSEGVVAFLILPKTKKDFFQSPPLHEPANMTTDPSSYYHTVTLNY (Bundibugyo ebolavirus); SEQ ID NO: 49:AFHKDGAFFLYDRLASTVIYRGVNFAEGVIAFLILAKPKETFLQS PPIREAVNYTENTSSYYATSYLEY(Sudan ebolavirus); SEQ ID NO: 50:AFHKEGAFFLYDRLASTIIYRGTTFAEGVIAFLILPKARKDFFQSP PLHEPANMTTDPSSYYHTTTINY(Tai Forest ebolavirus); or SEQ ID NO: 61:ALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMI FSRQGQGYRHMNLTSTNKYWTSSNGT(Marburg marburgvirus); or (E) an amino acid sequence set forth as(antigenic site IV.2, 286-296): SEQ ID NO: 51: GEWAFWETKKN (Zaireebolavirus Mayinga, Kikwit, Makona); SEQ ID NO: 52: GEWAFWENKKN(Bundibugyo ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus); or SEQID NO: 62: (Marburg marburgvirus); and wherein the peptide is no morethan 100 amino acids in length and induces a neutralizing immuneresponse to filovirus in a subject.
 9. The isolated peptide of claim 8,consisting essentially of or consisting of the amino acid sequence setforth as any one of SEQ ID NOs: 17-25, 29-31, 47-52, or 58-62.
 10. Theisolated peptide claim 1, wherein the peptide is no more than 50 aminoacids in length.
 11. The isolated peptide of claim 1, wherein thepeptide is no more than 40 amino acids in length.
 12. The isolatedpeptide of claim 1, linked to a heterologous carrier.
 13. The isolatedpeptide of claim 12, wherein the peptide is linked to the carrier by alinker.
 14. The isolated peptide of claim 12, wherein the carriercomprises Keyhole Limpet Hemocyanin (KLH), Concholepas ConcholepasHemocyanin (CCH), Ovalbumin (OVA), bovine serum albumin, recombinant B.anthracis protective antigen, recombinant P. aeruginosa exotoxin A,tetanus toxoid, tetanus toxin heavy chain C fragment, diphtheria toxoid,diphtheria toxin variant CRM197, pertussis toxoid, H influenza protein D(HiD), recombinant Clostridium difficile toxin B subunit (rBRU), C.perfringens toxoid, or analogs or mimetics of and combinations of two ormore thereof.
 15. The isolated peptide of claim 14, wherein the tetanustoxoid carrier is Keyhole Limpet Hemocyanin (KLH).
 16. The isolatedpeptide of claim 1, wherein the peptide induces a neutralizing immuneresponse to ebolavirus in the subject.
 17. An isolated nucleic acidmolecule encoding the peptide of claim
 1. 18. A vector comprising thenucleic acid molecule of claim 17 operably linked to a promoter.
 19. Thevector of claim 18, wherein the vector is an expression vector.
 20. Thevector of claim 18, wherein the vector is a viral vector.
 21. Animmunogenic composition comprising the peptide, nucleic acid molecule,or vector of claim 1, and a pharmaceutically acceptable carrier.
 22. Theimmunogenic composition of claim 21, further comprising an adjuvant. 23.The immunogenic composition of claim 21, comprising two or more of thepeptides, or one or more nucleic acid molecules or vectors encoding twoor more of the peptides.
 24. The immunogenic composition of claim 23,comprising: the peptide comprising, consisting essentially of, orconsisting of SEQ ID NO: 10; and the peptide comprising, consistingessentially of, or consisting of SEQ ID NO:
 3. 25. The immunogeniccomposition of claim 24, further comprising: the peptide comprising,consisting essentially of, or consisting of SEQ ID NO: 17; the peptidecomprising, consisting essentially of, or consisting of SEQ ID NO: 21;and the peptide comprising, consisting essentially of, or consisting ofSEQ ID NO:
 29. 26. A method for generating an immune response toebolavirus GP in a subject, comprising administering to the subject aneffective amount of the immunogenic composition of claim 21 to generatethe immune response.
 27. The method of claim 26, wherein the immuneresponse inhibits or treats an ebolavirus infection.
 28. A method ofdetecting a biological sample from a subject with a filovirus infectionor from a subject previously infected with a filovirus, comprising:contacting a blood or serum sample from the subject with an effectiveamount of the isolated peptide of claim 1 under conditions sufficient toform an immune complex between the peptide and antibodies in the bloodor serum sample; and detecting the presence of the immune complex,wherein the presence of the immune complex indicates that the biologicalsample is from a subject with the filovirus infection or from a subjectpreviously infected with the filovirus.
 29. The method of claim 28,wherein the filovirus is an ebolavirus.
 30. The method of claim 29,wherein detecting the presence of the immune complex in the biologicalsample indicates that the subject has an Ebola virus infection.
 31. Themethod of claim 26, wherein the ebolavirus is selected from any one ofZaire ebolavirus, Bundibugyo ebolavirus, Sudan ebolavirus, or Tai Forestebolavirus.
 32. (canceled)